Blockade of cd7 expression and chimeric antigen receptors for immunotherapy of t-cell malignancies

ABSTRACT

The present invention provides compositions comprising an anti-CD7 chimeric activating receptor (CAR) and an anti-CD7 protein expression blocker, and methods of using such compositions in cancer therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/821,153, filed Nov. 22, 2017, which claims the benefit of U.S.Provisional Application No. 62/425,398, filed on Nov. 22, 2016, and U.S.Provisional Application No. 62/543,696, filed Aug. 10, 2017, which areexpressly incorporated by reference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jul. 14, 2022, isnamed 62190_702_301_SL.xml and is 53,390 bytes in size.

BACKGROUND OF THE INVENTION

Chimeric antigen receptors (CARs) can redirect immune cells tospecifically recognize and kill tumor cells. CARs are artificialmulti-molecular proteins constituted by a single-chain variable region(scFv) of an antibody linked to a signaling molecule via a transmembranedomain. When the scFv ligates its cognate antigen, signal transductionis triggered, resulting in tumor cell killing by CAR-expressingcytotoxic T lymphocytes (Eshhar Z, Waks T, et al. PNAS USA.90(2):720-724, 1993; Geiger T L, et al. J Immunol. 162(10):5931-5939,1999; Brentjens R J, et al. Nat Med. 9(3):279-286, 2003; Cooper L J, etal. Blood 101(4):1637-1644, 2003; Imai C, et al. Leukemia. 18:676-684,2004). Clinical trials with CAR-expressing autologous T lymphocytes haveshown positive responses in patients with B-cell refractory leukemia andlymphoma (see, e.g., Till B G, et al. Blood 119(17):3940-3950, 2012;Maude S L, et al. N Engl J Med. 371(16):1507-1517, 2014).

The development of CAR technology to target T cell malignancies haslagged far behind the progress made for their B-cell counterparts. Noveltherapies for T-cell malignancies are needed but progress to date hasbeen slow. In particular, effective immunotherapeutic options arelacking and treatment of T-cell acute lymphocytic leukemia (T-ALL)relies on intensive chemotherapy and hematopoietic stem cell transplant.Despite the morbidity and mortality of these approaches, results are farfrom satisfactory.

In sum, there is a significant unmet need for new therapeutic optionsfor patients with T-cell malignancies.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an engineered immune cellcomprising: (i) a nucleic acid comprising a nucleotide sequence encodinga target-binding molecule linked to a localizing domain, wherein saidtarget-binding molecule is a first antibody that specifically binds toCD7; and (ii) a nucleic acid comprising a nucleotide sequence encoding achimeric antigen receptor (CAR), wherein said CAR comprises a 4-1BBintracellular signaling domain, a CD3ζ intracellular signaling domain,and a second antibody that specifically binds to CD7.

In some embodiments, the first antibody that specifically binds to CD7is a first single chain variable fragment (scFv). In certainembodiments, the second antibody that specifically binds to CD7 is asecond single chain variable fragment (scFv).

In some embodiments, the first single chain variable fragment (scFv)comprises a heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:1 and a light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:2. In other embodiments, the first single chainvariable fragment (scFv) comprises a heavy chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:14 and a light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:15. In certainembodiments, the first single chain variable fragment (scFv) comprises aheavy chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO:16 and a light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:17.

In some embodiments, the localizing domain comprising an amino acidsequence selected from the group consisting of an endoplasmic reticulum(ER) retention sequence, a Golgi retention sequence, a proteasomelocalizing sequence, and a transmembrane domain sequence derived fromCD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε,CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B. In someembodiments, the localizing domain comprises an endoplasmic reticulum(ER) retention sequence comprising an amino acid sequence of SEQ ID NO:8or SEQ ID NO:9. In other embodiments, the localizing domain comprisestransmembrane domain sequence derived from CD8a hinge and transmembranedomain sequence comprising an amino acid sequence of SEQ ID NO:13. Insome embodiments, proteasome localization of the target-binding molecule(e.g., scFv) is achieved by linking the scFv sequence to a tripartitemotif containing 21 (TRIM21) targeting domain sequence and coexpressinga nucleic acid sequence encoding the human TRIM21 E3 ubiquitin ligaseprotein.

In some embodiments, the 4-1BB intracellular signaling domain comprisesan amino acid sequence of SEQ ID NO:3 and wherein the CD3ζ intracellularsignaling domain comprises an amino acid sequence of SEQ ID NO:4.

In some embodiments, the hinge and transmembrane domain comprises anamino acid sequence of SEQ ID NO:10.

In some embodiments, the second single chain variable fragment (scFv)comprises a heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:1 and a light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:2. In other embodiments, the second single chainvariable fragment (scFv) comprises a heavy chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:14 and a light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:15. In yet otherembodiments, the second single chain variable fragment (scFv) comprisesa heavy chain variable domain having at least 90% sequence identity tothe amino acid sequence of SEQ ID NO:16 and a light chain variabledomain having at least 90% sequence identity to the amino acid sequenceof SEQ ID NO:17.

In some embodiments, the engineered cell is an engineered T cell, anengineered natural killer (NK) cell, an engineered NK/T cell, anengineered monocyte, an engineered macrophage, or an engineereddendritic cell.

In another aspect, the present invention provides an engineered immunecell comprising: (i) a target-binding molecule linked to a localizingdomain, wherein said target-binding molecule is a first antibody thatspecifically binds to CD7; and (ii) a chimeric antigen receptor (CAR),wherein said CAR comprises a 4-1BB intracellular signaling domain, aCD3ζ intracellular signaling domain, and a second antibody thatspecifically binds to CD7.

In some embodiments, the first antibody that specifically binds to CD7is a first single chain variable fragment (scFv). In certainembodiments, the second antibody that specifically binds to CD7 is asecond single chain variable fragment (scFv).

In some embodiments, the first single chain variable fragment (scFv)comprises a heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:1 and a light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:2. In other embodiments, the first single chainvariable fragment (scFv) comprises a heavy chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:14 and a light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:15. In certainembodiments, the first single chain variable fragment (scFv) comprises aheavy chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO:16 and a light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:17.

In some embodiments, the localizing domain comprising an amino acidsequence selected from the group consisting of an endoplasmic reticulum(ER) retention sequence, a Golgi retention sequence, a proteasomelocalizing sequence, and a transmembrane domain sequence derived fromCD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε,CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B. In someembodiments, the localizing domain comprises an endoplasmic reticulum(ER) retention sequence comprising an amino acid sequence of SEQ ID NO:8or SEQ ID NO:9. In other embodiments, the localizing domain comprisestransmembrane domain sequence derived from CD8a hinge and transmembranedomain sequence comprising an amino acid sequence of SEQ ID NO:13.

In some embodiments, the 4-1BB intracellular signaling domain comprisesan amino acid sequence of SEQ ID NO:3 and wherein the CD3ζ intracellularsignaling domain comprises an amino acid sequence of SEQ ID NO:4.

In some embodiments, the hinge and transmembrane domain comprises anamino acid sequence of SEQ ID NO:10.

In some embodiments, the second single chain variable fragment (scFv)comprises a heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:1 and a light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:2. In other embodiments, the second single chainvariable fragment (scFv) comprises a heavy chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:14 and a light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:15. In yet otherembodiments, the second single chain variable fragment (scFv) comprisesa heavy chain variable domain having at least 90% sequence identity tothe amino acid sequence of SEQ ID NO:16 and a light chain variabledomain having at least 90% sequence identity to the amino acid sequenceof SEQ ID NO:17.

In some embodiments, the engineered cell is an engineered T cell, anengineered natural killer (NK) cell, an engineered NK/T cell, anengineered monocyte, an engineered macrophage, or an engineereddendritic cell.

In some embodiments, provided herein is a pharmaceutical compositioncomprising the engineered immune cell described herein and apharmaceutically acceptable carrier.

In another aspect, the present invention provides methods of producingthe engineered immune cell described herein. The method can comprise:(i) introducing into an immune cell (a) a first nucleic acid comprisinga nucleotide sequence encoding a target-binding molecule linked to alocalizing domain, wherein said target-binding molecule is a firstantibody that specifically binds to CD7; and (b) a second nucleic acidcomprises a nucleotide sequence encoding a CAR, wherein said CARcomprises a 4-1BB intracellular signaling domain, a CD3ζ intracellularsignaling domain, and a second antibody that specifically binds to CD7;and (ii) isolating the engineered immune cell comprising saidtarget-binding molecule linked to said localizing domain and said CAR,thereby producing said engineered immune cell.

In yet another aspect, the present invention provides methods oftreating cancer in a subject (e.g., patient) in need thereof, comprisingadministering a therapeutic amount of an engineered immune cell to saidpatient, thereby treating cancer in the subject in need thereof. In someembodiments, engineered immune cell comprises: (i) a nucleic acidcomprising a nucleotide sequence encoding a target-binding moleculelinked to a localizing domain, wherein said target-binding molecule is afirst antibody that specifically binds to CD7; and (ii) a nucleic acidcomprising a nucleotide sequence encoding a chimeric antigen receptor(CAR), wherein said CAR comprises a 4-1BB intracellular signalingdomain, a CD3ζ intracellular signaling domain, and a second antibodythat specifically binds to CD7.

In some embodiments, the first antibody that specifically binds to CD7is a first single chain variable fragment (scFv). In certainembodiments, the second antibody that specifically binds to CD7 is asecond single chain variable fragment (scFv).

In some embodiments, the first single chain variable fragment (scFv)comprises a heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:1 and a light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:2. In other embodiments, the first single chainvariable fragment (scFv) comprises a heavy chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:14 and a light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:15. In certainembodiments, the first single chain variable fragment (scFv) comprises aheavy chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO:16 and a light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:17.

In some embodiments, the localizing domain comprising an amino acidsequence selected from the group consisting of an endoplasmic reticulum(ER) retention sequence, a Golgi retention sequence, a proteasomelocalizing sequence, and a transmembrane domain sequence derived fromCD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε,CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B. In someembodiments, the localizing domain comprises an endoplasmic reticulum(ER) retention sequence comprising an amino acid sequence of SEQ ID NO:8or SEQ ID NO:9. In other embodiments, the localizing domain comprisestransmembrane domain sequence derived from CD8a hinge and transmembranedomain sequence comprising an amino acid sequence of SEQ ID NO:13.

In some embodiments, the 4-1BB intracellular signaling domain comprisesan amino acid sequence of SEQ ID NO:3 and wherein the CD3ζ intracellularsignaling domain comprises an amino acid sequence of SEQ ID NO:4.

In some embodiments, the hinge and transmembrane domain comprises anamino acid sequence of SEQ ID NO:10.

In some embodiments, the second single chain variable fragment (scFv)comprises a heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:1 and a light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:2. In other embodiments, the second single chainvariable fragment (scFv) comprises a heavy chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:14 and a light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:15. In yet otherembodiments, the second single chain variable fragment (scFv) comprisesa heavy chain variable domain having at least 90% sequence identity tothe amino acid sequence of SEQ ID NO:16 and a light chain variabledomain having at least 90% sequence identity to the amino acid sequenceof SEQ ID NO:17.

In some embodiments, the engineered cell is an engineered T cell, anengineered natural killer (NK) cell, an engineered NK/T cell, anengineered monocyte, an engineered macrophage, or an engineereddendritic cell.

In some embodiments, the engineered immune cell is administered intosaid subject (e.g., patient) by intravenous infusion, intra-arterialinfusion, intraperitoneal infusion, direct injection into tumor and/orperfusion of tumor bed after surgery, implantation at a tumor site in anartificial scaffold, or intrathecal administration.

In some embodiments, the cancer is a T cell malignancy. In oneembodiment, the T cell malignancy is early T cell progenitor acutelymphoblastic leukemia (ETP-ALL).

The present disclosure provides engineered immune cells and methods ofuse thereof for treating T cell hematologic malignancies. One skilled inthe art recognizes that self-killing or fratricide of CAR T-cells andkilling of normal T cells can arise when CAR-T effector cells are usedto treat T cell leukemias. As such, there is a need for engineeredimmune cells and therapeutic methods that minimize or eliminate T cellfratricide.

The engineered immune cells and treatment methods described hereinutilize novel fratricide-resistant CAR-T cells, such as engineeredanti-CD7 PEBL and anti-CD7 CAR-T cells. The engineered immune cells canelicit potent and durable therapeutic effects in patients with T-cellmalignancies including relapsed T-cell malignancies. Such cells canresult in efficient targeting and killing of malignant T cells withoutsignificant effector T cell fratricide.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-FIG. 1D illustrate CD7 expression in T-ALL. Percentage of ALLcells expressing CD7 at diagnosis, relapse and during chemotherapy(MRD); the number of bone marrow samples studied at each stage is shown(FIG. 1A). CD7 mean fluorescence intensity (MFI) in T-ALL cells andresidual normal T-cells from the same samples (n=19; P<0.0001 by pairedt test) (FIG. 1B). CD7 MFI in T-ALL cells at diagnosis or relapse(“D/R”) and in follow-up bone marrow samples with MRD (n=18) (FIG. 1C).Flow cytometric contour plots illustrate CD7 expression in T-ALL cells(CD3-negative) and normal T cells (CD3-positive) at diagnosis, MRD, andrelapse in one representative patient (FIG. 1D).

FIG. 2A-FIG. 2E show the design, expression and signaling of theanti-CD7 CAR. Schema of the anti-CD7-41BB-CD3ζ construct (FIG. 2A). Flowcytometric analysis of Jurkat cells transduced with either GFP alone(“Mock”) or GFP plus anti-CD7 CAR. Dot plots illustrate GFPfluorescence, and CAR expression after staining with biotin-conjugatedgoat anti-mouse F(ab′)2 antibody and streptavidin-APC (JacksonImmunoResearch) (FIG. 2B). Western blot analysis of CAR expression inJurkat cells (FIG. 2C). Cell lysates of mck- and CAR-transduced Jurkatcells were separated on a 10% polyacrylamide gel under reducing ornon-reducing conditions. The blotted membrane was probed with mouseanti-human CD3ζ antibody (8D3; BD Biosciences) and goat anti-mouse IgGconjugated to horseradish peroxidase (R&D Systems). Antibody binding wasrevealed with Clarity Western ECL Substrate (Bio-Rad). Anti-CD7 CARinduces expression of activation markers upon ligation. Bars show mean(±SD) of CD25 and CD69 MFI in CAR- and mock-transduced Jurkat cellsafter 24 hours with or without CD7+ MOLT-4 cells. P values by t test areshown for significant differences (*=0.016; ***<0.001) (FIG. 2D). FIG.2E provides representative flow cytometric histograms of the experimentsshown in FIG. 2D.

FIG. 3A-FIG. 3I illustrate expression of anti-CD7 CAR in humanperipheral blood T-cells results in fratricide which is prevented by CD7downregulation. Percentage of viable T cells recovered 24 hours afterelectroporation with or without anti-CD7 CAR mRNA (n=7) (FIG. 3A).Viable cells were counted by flow cytometry. Percentage of viable Tcells recovered 24 hours after CAR transduction with a retroviral vectoras compared to cells from the same donors transduced with GFP alone(“Mock”) (n=10) (FIG. 3B). Percent of viable CAR- or mock-transduced Tcells recovered during the week following transduction (FIG. 3C). Shownare follow-up results for 5 of the 10 experiments shown in FIG. 3B.Percentage of CD107a in T cells after electroporation with or withoutanti-CD7 CAR mRNA (FIG. 3D). Mean (±SD) of triplicate measurements areshown. Schematic representation of anti-CD7 Protein Expression Blocker(PEBL) constructs (FIG. 3E). Representative flow cytometric histogramsillustrate CD7 expression in T-lymphocytes after retroviral transductionof 3 anti-CD7 PEBLs, or mock-transduced GFP alone (“Mock”) (FIG. 3F).T-cells were stained with anti-CD7-PE (M-T701; BD Biosciences).Percentage of CD7 expression in T cells retrovirally transduced with theanti-CD7 PEBL-1, or mock-transduced (n=5) (FIG. 3G). Flow cytometric dotplots illustrate downregulation of CD7 expression in T cells by PEBLtransduction, together with expression of anti-CD7-41BB-CD3ζ CAR 12hours after electroporation with or without CAR mRNA (FIG. 3H). Cellswere stained with biotin-conjugated goat anti-mouse F(ab′)2 antibody andstreptavidin-APC (Jackson ImmunoResearch). Percentage of viable T cellstransduced with anti-CD7 PEBL recovered 24 hours after electroporationof anti-CD7 CAR mRNA as compared to cells electroporated with theanti-CD7 CAR mRNA but transduced with a vector without anti-CD7 PEBL(n=6) (FIG. 3I). Number of viable cells was measured by flow cytometry.**, P<0.01; ***, P<0.001.

FIG. 4A-FIG. 4F show that CD7 downregulation by PEBL did not alterT-cell phenotype, proliferation and functionality. Percentage of CD4 andCD8 cells 7-14 days after retroviral transduction with either anti-CD7PEBL or GFP alone (“Mock”) (FIG. 4A). Each symbol corresponds to adifferent T cell donor. Growth rate of PEBL- and mock-transduced T cells(from 3 donors) maintained with 200 IU/mL IL-2 for 14 days (FIG. 4B).Symbols represent mean (±SD) of triplicate measurements. PEBL- andmock-transduced T cells were electroporated with eitheranti-CD19-41BB-CD3ζ CAR mRNA or no mRNA (FIG. 4C). Flow cytometric dotplots illustrate GFP and CAR expression 12 hours after electroporation.CAR was detected with biotin-conjugated goat anti-mouse F(ab′)2 antibodyand streptavidin-APC (Jackson ImmunoResearch). Cytotoxicity of PEBL- ormock-transduced T cells, electroporated with or without anti-CD19 CARmRNA, against CD19+ ALL cells (OP-1) (FIG. 4D). Bars show mean (±SD) of4-hour cytotoxicity at a 1:1 E:T. FIG. 4E shows CD107a expression in Tcells from experiments identical to those described in FIG. 4D. FIG. 4Fshows IFNγ production in PEBL- or mock-transduced T cells,electroporated with or without anti-CD19 CAR mRNA, and co-cultured withOP-1 for 6 hours at E:T 1:1. Bars represent mean (±SD) of triplicateexperiments. ***, P<0.001; ****, P<0.0001.

FIG. 5A-FIG. 5F show T cells with downregulated CD7 by PEBL acquirepowerful cytotoxicity against CD7+ leukemic cells after expression ofanti-CD7 CAR. Cytotoxicity of anti-CD7 PEBL-transduced T-cellselectroporated with or without anti-CD7 CAR mRNA against CD7+ cell lines(FIG. 5A). Shown are data for 4-hour assays at 1:1 E:T. Symbols indicatethe mean of 3 measurements each with T cells from 4 donors for MOLT-4,CCRF-CEM and Jurkat, and 5 donors for Loucy and KG1a (P<0.001 for eachcomparison). Cytotoxicity of anti-CD7 PEBL-transduced T-cellselectroporated with or without anti-CD7 CAR mRNA against primaryleukemic cells from patients with T-ALL (FIG. 5B). Shown are data for4-hour assays at the indicated E:T. Symbols refer to mean (±SD) of 3measurements. FIG. 5C shows overall specific cytotoxicity of T-cellstransduced with either anti-CD7 PEBL or GFP alone (“Mock”), afterelectroporation with anti-CD7 CAR mRNA against the 5 CD7+ cell lines. Tcells from 3 donors were tested, at 1:1 E:T, in 4-hour assays. Eachsymbol represents specific percent cytotoxicity against CD7+ cell line,after subtraction of the percent cytotoxicity obtained with the same Tcells electroporated without mRNA. Horizontal bars indicate the medianfor each group. Anti-CD7 PEBL- or mock-transduced T-cells from 3 donorswere electroporated with or without anti-CD7 CAR mRNA (FIG. 5D).Cytotoxicity against MOLT-4 was tested at 1:1 E:T in 4-hour assays.Shown is the mean fluorescence intensity (MFI) of anti-CD107a-PE (H4A3;BD Biosciences). Bars represent mean (±SD) of triplicate experiments.Anti-CD7 PEBL-transduced T-cells were retrovirally transduced witheither anti-CD7 CAR or mock-transduced, and tested against primaryleukemic cells from patients with T-ALL (FIG. 5E). Each symbol representmean (±SD) of triplicate experiments. Mock- or PEBL-transduced T-cells,sequentially transduced with or without anti-CD7 CAR, were culturedalone or in presence of Streck-treated MOLT-4 cells, added weekly and120 IU/mL IL-2 (FIG. 5F). Symbols indicate mean (±SD) percentage of cellrecovery relative to number of input cells in triplicate cultures. **,P<0.01, ***, P<0.001; ****, P<0.0001.

FIG. 6A-FIG. 6D show PEBL-transduced T-cells expressinganti-CD7-41BB-CD3ζ CAR exert antitumor activity in xenografts.NOD-SCID-IL2RG null mice were infused intravenously (i.v.) with 1×10⁶CCRF-CEM cells labelled with luciferase. 2×10⁷ PEBL-CAR T cells wereadministered i.v. on day 7 (FIG. 6A), or on day 3 and day 7 (FIG. 6B)after leukemic cell infusion to 3 and 5 mice, respectively. Theremaining mice received either mock-transduced T cells, or RPMI-1640instead of cells (“Control”). All mice received 20,000 IU IL-2 onceevery two days intraperitoneally (i.p.). Shown is in vivo imaging ofleukemia cell growth after D-luciferin i.p. injection. Ventral images ofmice on day 3 in FIG. 6B are shown with enhanced sensitivity todemonstrate CCRF-CEM engraftment in all mice. The complete set ofluminescence images is in FIG. 14. FIG. 6C shows leukemia cell growth inmice shown in FIG. 6A and FIG. 6B expressed as photons per second. Eachsymbol corresponds to bioluminescence measurements in each mouse,normalised to the average of ventral plus dorsal signals in all micebefore CAR-T cell infusion. Kaplan-Meier curves show overall survival ofmice in the different groups (8 in each group) (FIG. 6D). Mice wereeuthanized when the total bioluminescence signal reached 1×10¹⁰ photonsper second. P values calculated by log-rank test.

FIG. 7A-FIG. 7E show PEBL-CAR-T cell activity against ETP-ALL in apatient-derived xenograft (PDX) model. Primary ETP-ALL cells, previouslypropagated in NOD-SCID-IL2RGnull mice, were infused intravenously (i.v.)in 10 NOD-SCID-IL2RGnull mice at 2×10⁶ cells per mouse (FIG. 7A). Fivemice (“Controls”) were left untreated. The remaining 5 mice received asingle i.v. infusion of PEBL-CAR T cells (2×10⁷ in PEBL-CAR #1, 2×10⁶ inthe remaining 4 mice) at the indicated time point (grey arrow), as wellas 20,000 IU IL-2 i.p. every two days; IL-2 was also administered to 2of the 5 control mice. Black symbols (left y axes) indicate the numberof ETP-ALL cells/mL counted in peripheral blood. Grey symbols (right yaxes) show numbers of PEBL-CAR T cells. Mice were euthanized when thepercentage of ETP-ALL cells among blood mononucleated cells reached≥80%. Percentage of ETP-ALL (denominator, total human plus mouse CD45+cells) in various organs of the 5 untreated mice (FIG. 7B). Blood smearsof treated (PEBL-CAR #1) and untreated ETP-ALL 7 days after infusion ofT cells; smudge cells were prominent in blood after PEBL-CAR T cells(FIG. 7C). Flow cytometric dot plots show the presence of CD7+ CD3−ETP-ALL cells in the tissues of an untreated control mouse with ETP-ALLand of CD7− CD3+ PEBL-CAR T cells in the PEBL-CAR #1 mouse treated withPEBL-CAR-T cells (FIG. 7D). No ETP-ALL (<0.01%) was detected in thetreated mouse. Events shown were normalized to the events acquired forthe corresponding plots shown in the control mouse. Spleen of treated(PEBL-CAR #1) and untreated mice (FIG. 7E).

FIG. 8A-FIG. 8C show specificity and function of theanti-CD7-41BB-CD3ζCAR. OP-1 (CD7−) and MOLT-4 (CD7+) were incubated withsupernatant collected from Jurkat cells transduced with anti-CD7 scFv,or transduced with a vector containing GFP only (“Control”) (FIG. 8A).After washing, cells were incubated with biotin-conjugated goatanti-mouse F(ab′)2 antibody followed by streptavidin-APC (JacksonImmunoResearch). Flow cytometric histograms illustrate binding of theanti-CD7 scFv to MOLT-4 but not OP-1. Jurkat cells were transduced withanti-CD7-41BB-CD3ζCAR, anti-CD19-41BB-CD3ζCAR, or a vector containingGFP alone (FIG. 8B). These cells were co-cultured at 1:1 E:T with theCD7+ MOLT-4 or CCRF-CEM cells, or with the CD7− cells OP-1. Target cellswere labelled with calcein red-orange AM (Invitrogen). After 30 minutesincubation, the percentage of cell doublets was measured by flowcytometry. Bars illustrate mean (±SD) of triplicate measurements. FIG.8C shows that CAR-mediated cell aggregation is inhibited bypre-incubating target cells with a soluble form of the anti-CD7 scFv.***P<0.001.

FIG. 9A and FIG. 9B show expression of anti-CD7-41BB-CD3ζCAR in humanperipheral blood T lymphocytes. FIG. 9A provides representative flowcytometric dot plots of T lymphocytes activated for 7 days withDynabeads Human T-Activator CD3/CD28 (ThermoFisher Scientific) and IL-2,and transduced with the anti-CD7 CAR. Flow cytometric dot plotsillustrate GFP fluorescence and CAR expression, the latter revealed bystaining with biotin-conjugated goat anti-mouse F(ab′)2 antibodyfollowed by streptavidin-APC (Jackson ImmunoResearch). FIG. 9B showsWestern blot analysis of CAR expression. Cell lysates of mock- andCAR-transduced T cells were separated on a 10% polyacrylamide gel underreducing or non-reducing conditions. The blotted membrane was probedwith a mouse anti-human CD3ζ antibody (8D3; BD Biosciences) followed bygoat anti-mouse IgG conjugated to horseradish peroxidase (R&D Systems).Antibody binding was revealed with Clarity Western ECL Substrate(Bio-Rad).

FIG. 10A and FIG. 10B illustrate downregulation of CD7 proteinexpression with anti-CD7 PEBLs. Flow cytometric dot plot illustrate GFPexpression (x axes), CD7 expression (y axes, top row), and intracellularanti-CD7 PEBL-1 expression (y axes, bottom row) (FIG. 10A). Tlymphocytes were retrovirally transduced with anti-CD7 PEBL-1 or avector containing GFP alone (“Mock”). T-cells were stained with ananti-CD7 antibody (M-T701; BD Biosciences) conjugated to phycoerythrin.Intracellular expression of PEBL-1 was tested with a PE-conjugatedanti-Myc antibody (9B11; Cell Signaling Technology) which binds to thesequence EQKLISEEDL (SEQ ID NO:40) incorporated in the ER-binding motif.Prior to antibody labelling, cells were permeabilized with 8E reagent (apermeabilization reagent developed in our laboratory). FIG. 10B showsRT-PCR analysis of CD7 mRNA expression. cDNA derived from total mRNAextracted from Jurkat cells transduced with PEBL1-3, GFP alone (“mock”),or untransduced (“WT”) was used as template. CD7 cDNA (723 bp) wasamplified with the following primers: Forward, ATGGCCGGGCCTCCG (SEQ IDNO:38), Reverse, TCACTGGTACTGGTTGGG (SEQ ID NO:39). Electrophoresis wasperformed on a 1% agarose gel with SYBR Safe Gel Stain (ThermoFisher).No template control is also shown. A 87 bp (676-762th nucleotide) regionof glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified inparallel as a control.

FIG. 11A and FIG. 11B show that anti-CD7 CAR signal elicited highercytokine secretion in T cells with CD7 knock-down expression by anti-CD7PEBL. T lymphocytes from 3 donors were transduced with anti-CD7 PEBL orGFP alone (“Mock”) were electroporated with either anti-CD7-41BB-CD3ζmRNA or no mRNA. Intracellular IFNγ (FIG. 11A) and TNFα (FIG. 11B)expression in T cells after 6 hours of co-culture with MOLT4 wasmeasured. Bars represent mean (±SD) of triplicate MFI measurements. **,P<0.01; ***, P<0.001; ****, P<0.0001.

FIG. 12 shows that CD7-negative T-cells expressing anti-CD7-41BB-CD3ζCARexerted anti-tumour cytotoxicity against CD7+ cell lines. Shown areresults of 4-hour cytotoxicity assays performed with T cells transducedwith anti-CD7 PEBL and then transduced with either CD7-41BB-CD3ζ or GFPonly (“Mock”). Symbols represent mean (±SD) of triplicate experiments atthe indicated E:T ratios. P<0.001 for all comparisons.

FIG. 13A-FIG. 13E provide functional comparisons of anti-CD7-41BB-CD3ζand anti-CD19-41BB-CD3ζ CARs. FIG. 13A shows expression of anti-CD19 andanti-CD7 CARs (in an mCherry-containing vector) in peripheral blood Tcells previously transduced with anti-CD7 PEBL. Flow cytometry dot plotsillustrate mCherry expression and staining of T cells withbiotin-conjugated goat anti-mouse F(ab′)2 antibody followed bystreptavidin conjugated to allophycocyanin (Jackson ImmunoResearch).Results with T cell transduced with a vector containing mCherry alone(“Mock”) are also shown. Expression of CD19 in CCRF-CEM and Jurkat cellstransduced with a vector containing CD19 and GFP (FIG. 13B). CD19 wasdetected with anti-CD19 APC (Miltenyi Biotech). Four-hour cytotoxicityassays targeting CD19+ CCRF-CEM or CD19+ Jurkat cells with anti-CD19 oranti-CD7 PEBL-CAR-T cells at different E:T ratios (FIG. 13C). Symbolsindicate mean (±SD) of triplicate measurements. P<0.001 for data witheither CAR versus mock-transduced T cells at all E:T ratios. Long-termcytotoxicity of anti-CD19 or anti-CD7 PEBL-CAR-T cells at different E:Tratios as measured by live cell image analysis with IncuCyte Zoom System(Essen BioScience) (FIG. 13D). Symbols indicate mean (±SD) of 3measurements of CD19+ CCRF-CEM (top) or CD19+ Jurkat cells (bottom) inwells containing CAR-T cells, mock-transduced T cells, or no T cells.Measurements were performed at 4-hour intervals. Proliferative capacityof anti-CD19 and anti-CD7 PEBL-CAR-T cells with and without co-culturewith CD19+ Jurkat cells (FIG. 13E). Anti-CD7 PEBL-transduced T-cells,sequentially transduced with anti-CD19 or anti-CD7 CARs or mCherryalone, were cultured alone or in presence of irradiated CD19+ Jurkatcells, added weekly and 120 IU/mL IL-2. Symbols indicate mean (±SD)percentage of cell recovery relative to number of input cells intriplicate cultures.

FIG. 14A-FIG. 14C illustrate PEBL-transduced T-cells expressinganti-CD7-41BB-CD3ζ CAR exerted antitumor activity in mouse models.NOD-SCID-IL2RGnull mice were infused intravenously with 1×10⁶ CCRF-CEMcells labeled with luciferase. 2×10⁷ PEBL-CAR T cells were administeredintravenously on day 7 (FIG. 14A) or on day 3 and day 7 (FIG. 14B) afterleukemic cell infusion to 3 and 5 mice, respectively. The remaining micereceived either mock-transduced T cells, or RPMI-1640 instead of cells(“Control”). All mice received 20,000 IU IL-2 once every two daysintraperitoneally (i.p.). In vivo imaging of leukemia cell growth wasperformed after D-luciferin i.p. injection. Ventral images of mice onday 3 in FIG. 14B are shown with enhanced sensitivity to demonstrateleukemia cell engraftment in all mice. Leukemia cell growth expressed asphotons per second over time normalised to average of ventral plusdorsal signals in all mice before CAR-T cell infusion (FIG. 14C). Eachsymbol corresponds to bioluminescence measurements in each mouse.

FIG. 15A and FIG. 15B illustrate PEBL-transduced T cells expressinganti-CD7-41BB-CD3ζ CAR exerted antitumor activity in mouse models andremained active against cells collected at relapse. FIG. 15A showspercentage of CCRF-CEM cells among white blood cells in blood fromNOD-SCID-IL2RGnull mice infused i.v. with CCRF-CEM cells labelled withluciferase and then treated intravenously with eitherPEBL-CAR-transduced T-cells, mock-transduced T-cells, or RPMI-1640instead of cells (“Control”), as described for FIG. 6C. For “Control”and “Mock”, blood was obtained from euthanized mice that had reachedbioluminescence threshold of 10¹⁰ photons/second 17-23 days afterleukemia cells infusion. For PEBL-CAR mice, blood was obtained via cheekprick on day 24 after CCRF-CEM infusion. CCRF-CEM cells collected atrelapse from the spleen and liver of mice treated with PEBL-CAR werecultured for 2 days (FIG. 15B). They were then used as targets in 4-hourcytotoxicity assay at E:T 1:1 using PEBL-CAR- or mock-transduced T-cellsoriginally used for infusion. Comparison was also made with the samebatch of CCRF-CEM-expressing luciferase cells used to generate thexenograft. Percentage cytotoxicity was determined from platemeasurements of bioluminescence signal after addition of BrightGloluciferase assay system (Promega). Bars show mean (±SD) of triplicatemeasurements; each white and grey bar corresponds to cells from onemouse.

FIG. 16 provides immunophenotypic features of ETP-ALL at diagnosis andafter propagation in NOD-SCID-IL2RGnull mice. Flow cytometric contourplots show the immunophenotype diagnostic bone marrow samples of theETP-ALL used to develop the PDX model in this study and that of theETP-ALL cells recovered from the spleen of one of the control mice shownin FIG. 7. The following antibodies were used: CD7-PE, CD45-APC-H7,CD34-PerCP, CD8-BV510, CDS-PE-Cy7, CD3-PerCP (for cytoplasmic staining),CD3-V450 (for surface staining), all from BD Biosciences; CD33-BV421(Biolegend); CD1a-PE (Beckman Coulter). Quadrants were drawn based onstaining with isotype-matched non-reactive antibodies conjugated to thesame fluorochromes.

FIG. 17 provides a scheme of an exemplary embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The present invention is based, in part, on the design of a chimericantigen receptor (CAR) that is directed against CD7, a 40 kDa type Itransmembrane glycoprotein which is the primary marker for T cellmalignancies, and which is highly expressed in all cases of T cell ALL,including early T-cell progenitor acute lymphoblastic leukemia(ETP-ALL). As described herein, the anti-CD7 CAR induces T cells toexert specific cytotoxicity against T cell malignancies. Further, T cellcytotoxicity was shown to be markedly increased when anti-CD7 CAR wasused in combination with downregulation of CD7 expression on theeffector T cells. As demonstrated herein, downregulation (e.g.,elimination, reduction, and/or relocalization) of CD7 prevented thefratricidal effect exerted by the corresponding anti-CD7 CAR, allowinggreater T cell recovery after CAR expression as compared to cells thatretained the target antigen (e.g., CD7), and a more effectivecytotoxicity against T leukemia/lymphoma cells.

Accordingly, in one aspect, the present invention relates to anengineered immune cell comprising a nucleic acid that comprises anucleotide sequence encoding a chimeric antigen receptor (CAR), whereinthe CAR comprises intracellular signaling domains of 4-1BB and CD3ζ, andan antibody that specifically binds Cluster of Differentiation 7 (CD7).The CAR of the present invention is sometimes referred to herein as“anti-CD7-41BB-CD3ζ”. An exemplary embodiment is depicted in FIG. 17

As used herein, an “engineered” immune cell includes an immune cell thathas been genetically modified as compared to a naturally-occurringimmune cell. For example, an engineered T cell produced according to thepresent methods carries a nucleic acid comprising a nucleotide sequencethat does not naturally occur in a T cell from which it was derived.

In certain embodiments, the engineered immune cell is an engineered Tcell, an engineered natural killer (NK) cell, an engineered NK/T cell,an engineered monocyte, an engineered macrophage, or an engineereddendritic cell. In certain embodiments, the engineered immune cell is anengineered T cell. As used herein, the term “nucleic acid” refers to apolymer comprising multiple nucleotide monomers (e.g., ribonucleotidemonomers or deoxyribonucleotide monomers). “Nucleic acid” includes, forexample, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleicacid molecules can be naturally occurring, recombinant, or synthetic. Inaddition, nucleic acid molecules can be single-stranded, double-strandedor triple-stranded. In certain embodiments, nucleic acid molecules canbe modified. In the case of a double-stranded polymer, “nucleic acid”can refer to either or both strands of the molecule.

The term “nucleotide sequence,” in reference to a nucleic acid, refersto a contiguous series of nucleotides that are joined by covalentlinkages, such as phosphorus linkages (e.g., phosphodiester, alkyl andaryl-phosphonate, phosphorothioate, phosphotriester bonds), and/ornon-phosphorus linkages (e.g., peptide and/or sulfamate bonds). Incertain embodiments, the nucleotide sequence encoding, e.g., atarget-binding molecule linked to a localizing domain is a heterologoussequence (e.g., a gene that is of a different species or cell typeorigin).

The terms “nucleotide” and “nucleotide monomer” refer to naturallyoccurring ribonucleotide or deoxyribonucleotide monomers, as well asnon-naturally occurring derivatives and analogs thereof. Accordingly,nucleotides can include, for example, nucleotides comprising naturallyoccurring bases (e.g., adenosine, thymidine, guanosine, cytidine,uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, ordeoxycytidine) and nucleotides comprising modified bases known in theart.

As will be appreciated by those of skill in the art, in some aspects,the nucleic acid further comprises a plasmid sequence. The plasmidsequence can include, for example, one or more sequences of a promotersequence, a selection marker sequence, or a locus-targeting sequence.

As used herein, “antibody” means an intact antibody or antigen-bindingfragment of an antibody, including an intact antibody or antigen-bindingfragment that has been modified or engineered, or that is a humanantibody. Examples of antibodies that have been modified or engineeredare chimeric antibodies, humanized antibodies, multiparatopic antibodies(e.g., biparatopic antibodies), and multispecific antibodies (e.g.,bispecific antibodies). Examples of antigen-binding fragments includeFab, Fab′, F(ab′)₂, Fv, single chain antibodies (e.g., scFv), minibodiesand diabodies.

The term “specifically (or selectively) binds” or “specifically (orselectively) immunoreactive with,” when referring to a protein orpeptide, refers to a binding reaction that is determinative of thepresence of the protein, often in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and more typically more than 10 to 100 times background.Specific binding to an antibody under such conditions requires anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies can be selected to obtain only thosepolyclonal antibodies that are specifically immunoreactive with theselected antigen and not with other proteins. This selection may beachieved by subtracting out antibodies that cross-react with othermolecules. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Using Antibodies, A Laboratory Manual (1998) for a descriptionof immunoassay formats and conditions that can be used to determinespecific immunoreactivity).

In certain embodiments, the antibody that binds CD7 is a single-chainvariable fragment antibody (“scFv antibody”). scFv refers to antibodyfragments comprising the VH and VL domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. For a review of scFv, see Pluckthun (1994) The Pharmacology OfMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315. See also, PCT Publication No. WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. As would beappreciated by those of skill in the art, various suitable linkers canbe designed and tested for optimal function, as provided in the art, andas disclosed herein.

In certain embodiments, the anti-CD7 scFv comprises a variable heavychain (heavy chain variable region or VH) and a variable light chain(light chain variable region or VL) having an amino acid sequence thateach have at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to the VH and VL sequences set forthin SEQ ID NO: 1 and 2, respectively. The heavy chain variable region cancomprise at least 90% sequence identity, at least 91% sequence identity,at least 92% sequence identity, at least 93% sequence identity, at least94% sequence identity, at least 95% sequence identity, at least 96%sequence identity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VH sequence of SEQ ID NO:1. The light chain variable region cancomprise at least 90% sequence identity, at least 91% sequence identity,at least 92% sequence identity, at least 93% sequence identity, at least94% sequence identity, at least 95% sequence identity, at least 96%sequence identity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VL sequence of SEQ ID NO:2. In some instances, the heavy chainvariable region comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more) amino acid substitution in the sequence set forth in SEQ IDNO:1. In certain instances, the heavy chain variable region comprise 10or fewer amino acid (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)substitutions in the sequence set forth in SEQ ID NO:1. In someinstances, the light chain variable region comprise at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in thesequence set forth in SEQ ID NO:2. In certain instances, the light chainvariable region comprise 10 or fewer amino acid (e.g., 0, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10) substitutions in the sequence set forth in SEQ IDNO:2. In some embodiments, a nucleic acid sequence encoding a VHcomprises at least 90% sequence identity, at least 91% sequenceidentity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to the nucleic acid sequence setforth in SEQ ID NO:23. In other embodiments, a nucleic acid sequenceencoding a VL comprises at least 90% sequence identity, at least 91%sequence identity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to the nucleic acid sequence setforth in SEQ ID NO:24.

In certain embodiments, the anti-CD7 scFv comprises a variable heavychain (heavy chain variable region or VH) and a variable light chain(light chain variable region or VL) having a sequence that each have atleast 90% sequence identity, at least 91% sequence identity, at least92% sequence identity, at least 93% sequence identity, at least 94%sequence identity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VH and VL sequences set forth in SEQ ID NO: 14 and 15, respectively.The heavy chain variable region can comprise at least 90% sequenceidentity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VH sequence of SEQ ID NO:14. The light chain variable region cancomprise at least 90% sequence identity, at least 91% sequence identity,at least 92% sequence identity, at least 93% sequence identity, at least94% sequence identity, at least 95% sequence identity, at least 96%sequence identity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VL sequence of SEQ ID NO:15.

In some instances, the heavy chain variable region comprise at least one(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitutionin the sequence set forth in SEQ ID NO:14. In certain instances, theheavy chain variable region comprise 10 or fewer amino acid (e.g., 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions in the sequence set forth inSEQ ID NO:14. In some cases, the light chain variable region comprise atleast one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) amino acidsubstitution in the sequence set forth in SEQ ID NO:15. In certaincases, the heavy chain variable region comprise 10 or fewer amino acid(e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions in the sequenceset forth in SEQ ID NO:15.

In some embodiments, a nucleic acid sequence encoding a VH comprises atleast 90% sequence identity, at least 91% sequence identity, at least92% sequence identity, at least 93% sequence identity, at least 94%sequence identity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe nucleic acid sequence set forth in SEQ ID NO:25. In otherembodiments, a nucleic acid sequence encoding a VL comprises at least90% sequence identity, at least 91% sequence identity, at least 92%sequence identity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe nucleic acid sequence set forth in SEQ ID NO:26.

In certain embodiments, the anti-CD7 scFv comprises a variable heavychain (heavy chain variable region or VH) and a variable light chain(light chain variable region or VL) having a sequence that each have atleast 90% sequence identity, at least 91% sequence identity, at least92% sequence identity, at least 93% sequence identity, at least 94%sequence identity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VH and VL sequences set forth in SEQ ID NO: 16 and 17, respectively.The heavy chain variable region can comprise at least 90% sequenceidentity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VH sequence of SEQ ID NO:16. The light chain variable region cancomprise at least 90% sequence identity, at least 91% sequence identity,at least 92% sequence identity, at least 93% sequence identity, at least94% sequence identity, at least 95% sequence identity, at least 96%sequence identity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VL sequence of SEQ ID NO:17.

In some instances, the heavy chain variable region comprise at least one(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitutionin the sequence set forth in SEQ ID NO:16. In certain instances, theheavy chain variable region comprise 13 or fewer amino acid (e.g., 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) substitutions in the sequenceset forth in SEQ ID NO:16. In some cases, the light chain variableregion comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) amino acid substitution in the sequence set forth in SEQ ID NO:17.In certain cases, the heavy chain variable region comprise 5 or feweramino acid (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) substitutionsin the sequence set forth in SEQ ID NO:17. In some embodiments, anucleic acid sequence encoding a VH comprises at least 90% sequenceidentity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to the nucleic acid sequence setforth in SEQ ID NO:27. In other embodiments, a nucleic acid sequenceencoding a VL comprises at least 90% sequence identity, at least 91%sequence identity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe nucleic acid sequence set forth in SEQ ID NO:28.

In some embodiments, the scFv of the present invention comprises avariable heavy chain sequence having at least 90% sequence identity, atleast 91% sequence identity, at least 92% sequence identity, at least93% sequence identity, at least 94% sequence identity, at least 95%sequence identity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity to avariable heavy chain sequence of an anti-CD7 antibody. In someembodiments, the scFv of the present invention comprises a variablelight chain sequence having at least 90% sequence identity, at least 91%sequence identity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity to avariable light chain sequence of an anti-CD7 antibody. For instance, theanti-CD7 antibody can be any such recognized by one skilled in the art.

TABLE 1 Amino acid sequences of VH regions and VL regions of anti-CD7 scFvs Component Amino Acid Sequence TH69 VHEVQLVESGGGLVKPGGSLKLSCAASGLTFSS YAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMY YCARDEVRGYLDVWGAGTTVTVSS  (SEQ ID NO: 1)VL AAYKDIQMTQTTSSLSASLGDRVTISCSASQ GISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQ QYSKLPYTFGGGTKLEIKR  (SEQ ID NO: 2) 3a1fVH QVQLQESGAELVKPGASVKLSCKASGYTFTS YWMHWVKQRPGQGLEWIGKINPSNGRTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAV YYCARGGVYYDLYYYALDYWGQGTTVTVSS (SEQ ID NO: 14) VL DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRF SGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKR (SEQ ID NO: 15) T3- VH DVQLVESGGGLVQPGGSRKLSCAASGFTFSS3A1 FGMHWVRQAPEKGLEWVAYISSGSSTLHYAD TVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARWGNYPHYAMDYWGQGTSVTVSS  (SEQ ID NO: 16) VLDIVMTQSPASLAVSLGQRATISCRASKSVSA SGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAVTYYCQ HSRELPYTFGGGTKLEIK  (SEQ ID NO: 17)

TABLE 2 Nucleic acid sequences of VH regionsand VL regions of anti-CD7 scFvs Component Nucleic Acid Sequence TH69 VHGAGGTGCAGCTGGTCGAATCTGGAGGAGG ACTGGTGAAGCCAGGAGGATCTCTGAAACTGAGTTGTGCCGCTTCAGGCCTGACCTTC TCAAGCTACGCCATGAGCTGGGTGCGACAGACACCTGAGAAGCGGCTGGAATGGGTCG CTAGCATCTCCTCTGGCGGGTTCACATACTATCCAGACTCCGTGAAAGGCAGATTTAC TATCTCTCGGGATAACGCAAGAAATATTCTGTACCTGCAGATGAGTTCACTGAGGAGC GAGGACACCGCAATGTACTATTGTGCCAGGGACGAAGTGCGCGGCTATCTGGATGTCT GGGGAGCTGGCACTACCGTCACCGTCTCCAGC (SEQ ID NO: 23) VL GCCGCATACAAGGATATTCAGATGACTCAGACCACAAGCTCCCTGAGCGCCTCCCTGG GAGACCGAGTGACAATCTCTTGCAGTGCATCACAGGGAATTAGCAACTACCTGAATTG GTATCAGCAGAAGCCAGATGGCACTGTGAAACTGCTGATCTACTATACCTCTAGTCTG CACAGTGGGGTCCCCTCACGATTCAGCGGATCCGGCTCTGGGACAGACTACAGCCTGA CTATCTCCAACCTGGAGCCCGAAGATATTGCCACCTACTATTGCCAGCAGTACTCCAA GCTGCCTTATACCTTTGGCGGGGGAACAAAGCTGGAGATTAAAAGG (SEQ ID NO: 24) 3a1f VH CAGGTCCAGCTGCAGGAGTCAGGAGCTGAGCTGGTGAAGCCAGGGGCAAGCGTCAAAC TGTCCTGCAAGGCCTCTGGATATACATTCACTAGCTACTGGATGCACTGGGTGAAACA GAGACCCGGACAGGGCCTGGAGTGGATCGGAAAGATTAACCCTAGCAATGGCAGGACC AACTACAACGAAAAGTTTAAATCCAAGGCAACCCTGACAGTGGACAAGAGCTCCTCTA CAGCCTACATGCAGCTGAGTTCACTGACTTCAGAGGATAGCGCAGTGTACTATTGCGC CAGAGGCGGGGTCTACTATGACCTGTACTATTACGCCCTGGATTATTGGGGGCAGGGA ACCACAGTGACTGTCAGCTCC  (SEQ ID NO: 25) VLGACATCGAGCTGACCCAGAGTCCTGCTAC ACTGAGCGTGACTCCAGGCGATTCTGTCAGTCTGTCATGTCGGGCAAGCCAGTCCATC TCTAACAATCTGCACTGGTACCAGCAGAAATCCCATGAATCTCCACGACTGCTGATTA AGAGTGCCTCACAGAGCATCTCCGGCATTCCCTCCCGGTTCTCTGGCAGTGGGTCAGG AACTGACTTTACCCTGAGTATTAACTCAGTGGAGACAGAAGATTTCGGCATGTATTTT TGCCAGCAGAGCAATTCCTGGCCCTACACTTTCGGAGGCGGGACCAAACTGGAGATCA AGCGG (SEQ ID NO: 26) T3- VHGATGTGCAGCTGGTGGAGTCTGGGGGAGG 3A1 CTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTC AGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCG CATACATTAGTAGTGGCAGTAGTACCCTCCACTATGCAGACACAGTGAAGGGCCGATT CACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGG TCTGAGGACACGGCCATGTATTACTGTGCAAGATGGGGTAACTACCCTCACTATGCTA TGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 27) VL GACATTGTGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCA CCATCTCATGCAGGGCCAGCAAAAGTGTCAGTGCATCTGGCTATAGTTATATGCACTG GTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTA GAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCA ACATCCATCCTGTGGAGGAGGAGGATGCTGTAACCTATTACTGTCAGCACAGTAGGGA GCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA  (SEQ ID NO: 28)

The term “sequence identity” means that two nucleotide sequences or twoamino acid sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least, e.g., 70%sequence identity, or at least 80% sequence identity, or at least 85%sequence identity, or at least 90% sequence identity, or at least 95%sequence identity or more. For sequence comparison, typically onesequence acts as a reference sequence (e.g., parent sequence), to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

As those skilled in the art would appreciate, in certain embodiments,any of the sequences of the various components disclosed herein (e.g.,scFv, intracellular signaling domain, hinge, linker, localizingsequences, and combinations thereof) can have at least 90% sequenceidentity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe specific corresponding sequences disclosed herein. For example, incertain embodiments, the intracellular signaling domain 4-1BB can haveat least 90% sequence identity, at least 91% sequence identity, at least92% sequence identity, at least 93% sequence identity, at least 94%sequence identity, at least 95% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to SEQ ID NO:3, as long as itpossesses the desired function. In certain embodiments, theintracellular signaling domain of 4-1BB comprises the sequence set forthin SEQ ID NO:3 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

As another example, in certain embodiments, the intracellular signalingdomain 4-1BB can be replaced by another intracellular signaling domainfrom a co-stimulatory molecule such as CD28, OX40, ICOS, CD27, GITR,HVEM, TIM1, LFA1, or CD2. In some embodiments, the intracellularsignaling domain of the CAR can have at least 90% sequence identity, atleast 91% sequence identity, at least 92% sequence identity, at least93% sequence identity, at least 94% sequence identity, at least 95%sequence identity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to the intracellular signalingdomain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2.

As another example, in certain instances, the intracellular signalingdomain of 4-1BB can also include another intracellular signaling domain(or a portion thereof) from a co-stimulatory molecule such as CD28,OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2. In some embodiments,the additional intracellular signaling domain can have at least 90%sequence identity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR,HVEM, TIM1, LFA1, or CD2. In other embodiments, the additionalintracellular signaling domain comprises at least 90% sequence identity,at least 91% sequence identity, at least 92% sequence identity, at least93% sequence identity, at least 94% sequence identity, at least 95%sequence identity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to one or more intracellularsignaling domain fragment(s) of CD28, OX40, ICOS, CD27, GITR, HVEM,TIM1, LFA1, or CD2.

As another example, in certain embodiments, the intracellular signalingdomain CD3 can have at least 90% sequence identity, at least 91%sequence identity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to SEQ ID NO:4, as long as itpossesses the desired function. In certain embodiments, theintracellular signaling domain of CD3ζ comprises the sequence set forthin SEQ ID NO:4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

In some instances, the intracellular signaling domain comprises animmunoreceptor tyrosine-based activation motif (ITAM) or a portionthereof, as long as it possess the desired function. The intracellularsignaling domain of the CAR can include a sequence having at least 90%sequence identity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity toan ITAM. In certain embodiments, the intracellular signaling domain canhave at least 95% sequence identity, at least 96% sequence identity, atleast 97% sequence identity, at least 98% sequence identity, at least99% sequence identity, or 100% sequence identity to FcεRIγ, CD4, CD7,CD8, CD28, OX40 or H2-Kb, as long as it possesses the desired function.

In certain embodiments, the anti-CD7 CAR further comprises a hinge andtransmembrane sequence. Hinge and transmembrane sequences suitable foruse in the present invention are known in the art, and provided in,e.g., publication WO2016/126213, incorporated by reference in itsentirety. In certain embodiments, the hinge sequence comprises thesequence set forth in SEQ ID NO:5(TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD). In certain embodiments,the transmembrane sequence comprises the sequence set forth in SEQ IDNO:6 (IYIWAPLAGTCGVLLLSLVITLYC). In some embodiments, the hinge andtransmembrane domain of the anti-CD7 CAR can be include a signalingdomain (e.g., transmembrane domain) from CD8β, 4-1BB, CD28, CD34, CD4,FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2,FAS, FGFR2B, or another transmembrane protein.

In certain embodiments, the anti-CD7 CAR further comprises a CD8a signalpeptide (MALPVTALLLPLALLLHAARP; SEQ ID NO:7). A schematic of theanti-CD7 CAR comprising the embodiments described herein is shown inFIG. 17.

In certain aspects of the present invention, the chimeric antigenreceptor (CAR) can bind to a molecule that is expressed on the surfaceof a cell including, but not limited to members of the CD1 family ofglycoproteins, CD2, CD3ζ, CD4, CD5, CD7, CD8, CD25, CD28, CD30, CD38,CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, and CD137.

As described herein, T cell cytotoxicity was shown to be markedlyincreased when anti-CD7 CAR was used in combination with downregulationof CD7 expression on the effector T cells. As demonstrated herein,downregulation (e.g., elimination, reduction, and/or relocalization) ofCD7 prevented the fratricidal effect exerted by the correspondinganti-CD7 CAR, allowing greater T cell recovery after CAR expression ascompared to cells that retained the target antigen (e.g., CD7), and amore effective cytotoxicity against T leukemia/lymphoma cells. As thoseof skill in the art would appreciate, downregulation of CD7 expressionon the effector T cells can be achieved according to a variety of knownmethods including, for example, “intrabodies” against CD7 (as describedin WO2016/126213), RNAi against CD7, or gene editing methods such as,e.g., meganucleases, TALEN, CRISPR/Cas9, and zinc finger nucleases.

In certain embodiments, the engineered immune cell further comprises anucleic acid that comprises a nucleotide sequence encoding atarget-binding molecule linked to a localizing domain. The“target-binding molecule linked to a localizing domain” is sometimesreferred to herein as a protein expression blocker (PEBL) or in somecases, an “intrabody”, as described in WO2016/126213, the teachings ofwhich are incorporated by reference in their entirety. Exemplaryembodiments of a PEBL are shown in FIG. 3E and FIG. 17.

As used herein, “linked” in the context of the protein expressionblocker refers to a gene encoding a target-binding molecule directly inframe (e.g., without a linker) adjacent to one or more genes encodingone or more localizing domains. Alternatively, the gene encoding atarget-binding molecule may be connected to one or more gene encodingone or more localizing domains through a linker sequence, e.g., asdescribed in WO2016/126213. As would be appreciated by those of skill inthe art, such linker sequences as well as variants of such linkersequences are known in the art. Methods of designing constructs thatincorporate linker sequences as well as methods of assessingfunctionality are readily available to those of skill in the art.

In certain embodiments, the target-binding molecule is an antibody thatbinds CD7. In certain embodiments, the antibody is a scFv. In certainembodiments, the scFv comprises a VH sequence set forth in SEQ ID NO:1and a VL sequence set forth in SEQ ID NO:2. In certain embodiments, thescFv comprises a VH sequence set forth in SEQ ID NO:14 and a VL sequenceset forth in SEQ ID NO:15. In certain embodiments, the scFv comprises aVH sequence set forth in SEQ ID NO:16 and a VL sequence set forth in SEQID NO:17. As described herein, in certain embodiments, the scFvcomprises a VH and a VL having sequence that each have at least 90%sequence identity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VH and VL sequences set forth in SEQ ID NO: 1 and 2, respectively;SEQ ID NO:14 and SEQ ID NO:15, respectively; or SEQ ID NO:16 and SEQ IDNO:17, respectively.

In some embodiments, the nucleic acid sequence of SEQ ID NO:23 encodingan immunoglobulin heavy chain variable region of an anti-CD7 scFv andthe nucleic acid sequence of SEQ ID NO:24 encoding an immunoglobulinlight chain variable region of an anti-CD7 scFv is used to produce ananti-CD7 protein expression blocker. In other embodiments, the nucleicacid sequence of SEQ ID NO:25 encoding an immunoglobulin heavy chainvariable region of an anti-CD7 scFv and the nucleic acid sequence of SEQID NO:26 encoding an immunoglobulin light chain variable region of ananti-CD7 scFv is used to produce an anti-CD7 protein expression blocker.In certain embodiments, the nucleic acid sequence of SEQ ID NO:27encoding an immunoglobulin heavy chain variable region of an anti-CD7scFv and the nucleic acid sequence of SEQ ID NO:28 encoding animmunoglobulin light chain variable region of an anti-CD7 scFv is usedto produce an anti-CD7 protein expression blocker.

In certain embodiments, the antibody that binds CD7 in the context ofthe CAR, as described herein, can be different from the antibody thatbinds CD7 in the context of the target-binding molecule (the PEBL).Merely to illustrate, the antibody that binds CD7 in the context of theCAR can comprise a VH sequence set forth in SEQ ID NO:1 and a VLsequence set forth in SEQ ID NO:2, whereas the antibody that binds CD7in the context of the PEBL can comprise a VH sequence set forth in SEQID NO:14 and a VL sequence set forth in SEQ ID NO:15. In certainembodiments, the antibody that binds CD7 in the context of the CAR, asdescribed herein, can be the same as the antibody that binds CD7 in thecontext of the target-binding molecule (the PEBL).

In certain embodiments, the localizing domain of the PEBL comprises anendoplasmic reticulum (ER) or Golgi retention sequence; a proteosomelocalizing sequence; a transmembrane domain sequence derived from CD8α,CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ,CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B. In certain embodiments,the localizing domain comprises endoplasmic reticulum (ER) retentionpeptides EQKLISEEDLKDEL (SEQ ID NO:8), (GGGGS)₄AEKDEL (SEQ ID NO:9), orCD8α hinge and transmembrane domain(TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLY)(SEQ ID NO:10) followed by KYKSRRSFIDEKKMP (SEQ ID NO:11), as describedherein. The localizing domain can direct the PEBL to a specific cellularcompartment, such as the Golgi or endoplasmic reticulum, the proteasome,or the cell membrane, depending on the application. The ER or Golgiretention sequence comprises the amino acid sequence KDEL (SEQ IDNO:18); KKXX where X is any amino acid; KXD/E (such as KXD or KXE) whereX is any amino acid; or YQRL (SEQ ID NO:21). The proteasome localizingsequence can comprise a PEST (SEQ ID NO:22) motif.

In some embodiments, proteasome localization is achieved by linking thescFv sequence to a tripartite motif containing 21 (TRIM21) targetingdomain sequence and coexpressing the sequence encoding the human TRIM21E3 ubiquitin ligase protein. TRIM21 binds with high affinity to the Fcdomains of antibodies and can recruit the ubiquitin-proteosome complexto degrade molecules (e.g., proteins and peptides) bound to theantibodies. The TRIM21 targeting domain sequence encodes amino acidsequences selected from the group of human immunoglobulin G (IgG)constant regions (Fc) genes such as IgG1, IgG2, or IgG4 and is used toform a fusion protein comprising scFv and Fc domains. In thisembodiment, the exogenously expressed TRIM21 protein binds the scFv-Fcfusion protein bound to the target protein (e.g., CD7) and directs thecomplex to the proteasome for degradation.

Details of the amino acid sequence of the human TRIM21 E3 ligase proteincan be found, for example, in NCBI Protein database under NCBI Ref. Seq.No. NP_003132.2. Details of the nucleib acid sequence encoding the humanTRIM21 E3 ligase protein can be found, for example, in NCBI Proteindatabase under NCBI Ref. Seq. No. NM_003141.3.

In certain embodiments, the protein expression blocker is any one ormore of the anti-CD7 PEBL as disclosed in WO2016/126213, the disclosureis herein incorporated by reference in its entirety for all purposes.Accordingly, the engineered immune cells described herein can comprisean PEBL (a target-binding molecule linked to a localizing domain) thatbinds to CD7, as described in WO2016/126213. The sequences of thecomponents of anti-CD7 intrabodies as described in FIG. 2, and Tables 1and 2 of WO2016/126213. Exemplary embodiments of an anti-CD7 PEBL aredepicted in FIG. 3E and FIG. 17.

TABLE 3 Amino acid sequence information forselect components of anti-CD7 PEBLs Component SequenceCD8α signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 7) VH-VL linkerGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 12) CD8α hinge andTTTPAPRPPTPAPTIASQPLS transmembrane domain LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLY (SEQ ID NO: 10) Localization domainEQKLISEEDLKDEL  KDEL tethered to scFv (SEQ ID NO: 8)with myc (“myc KDEL”) Localization domain (GGGGS)₄AEKDEL “link.(20)AEKDEL” (SEQ ID NO: 9) Localization domainTTTPAPRPPTPAPTIASQPLS “mb DEKKMP” LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYKYKSRRSFIDEKKMP  (SEQ ID NO: 13)Anti-CD7 scFv VH  EVQLVESGGGLVKPGGSLKLS domain (TH69)CAASGLTFSSYAMSWVRQTPE KRLEWVASISSGGFTYYPDSV KGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLD VWGAGTTVTVSS  (SEQ ID NO: 1) Anti-CD7 scFv VL AAYKDIQMTQTTSSLSASLGD domain (TH69) RVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVP SRFSGSGSGTDYSLTISNLEP EDIATYYCQQYSKLPYTFGGGTKLEIKR (SEQ ID NO: 2)

In some embodiments, the anti-CD7 protein expression blocker comprisesan amino acid sequence of SEQ ID NO:1, an amino acid sequence of SEQ IDNO:2, and a VH-VL linker. The VH-VL linker can be a (GGGGS)_(n) linkerwhere n can range from 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. In oneembodiment, the anti-CD7 protein expression blocker comprises an aminoacid sequence of SEQ ID NO:1, an amino acid sequence of SEQ ID NO:2, andan amino acid sequence of SEQ ID NO:12. In some embodiments, theanti-CD7 protein expression blocker comprises an amino acid sequencehaving at least 90% sequence identity or at least 95% sequence identityto SEQ ID NO:1, the amino acid sequence of SEQ ID NO:2, and the aminoacid sequence of SEQ ID NO:12. In certain embodiments, the anti-CD7protein expression blocker comprises an amino acid sequence of SEQ IDNO:1, an amino acid sequence having at least 90% sequence identity or atleast 95% sequence identity to SEQ ID NO: 2, and an amino acid sequenceof SEQ ID NO:12. In other embodiments, the anti-CD7 protein expressionblocker comprises an amino acid sequence having at least 90% sequenceidentity or at least 95% sequence identity to SEQ ID NO:1, an amino acidsequence having at least 90% sequence identity or at least 95% sequenceidentity to SEQ ID NO:2, and an amino acid sequence of SEQ ID NO:12. Insome instance, the anti-CD7 protein expression blocker also comprises alocalization domain selected from any one sequence set forth in SEQ IDNO:8, SEQ ID NO:9, or SEQ ID NO:13. In some cases, the anti-CD7 proteinexpression blocker also comprises a CD8α signal peptide such as but notlimited to the CD8α signal peptide set forth in SEQ ID NO:7. In othercases, the anti-CD7 protein expression blocker also comprises a CD8αhinge and transmembrane domain such as but not limited to the CD8α hingeand transmembrane domain set forth in SEQ ID NO:10.

In some embodiments, the anti-CD7 protein expression blocker comprisesan amino acid sequence of SEQ ID NO:14, an amino acid sequence of SEQ IDNO:15, and a VH-VL linker. The VH-VL linker can be a (GGGGS)_(n) linkerwhere n can range from 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. In oneembodiment, the anti-CD7 protein expression blocker comprises an aminoacid sequence of SEQ ID NO:14, an amino acid sequence of SEQ ID NO:15,and an amino acid sequence of SEQ ID NO:12. In some embodiments, theanti-CD7 protein expression blocker comprises an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:14, the amino acidsequence of SEQ ID NO:15, and the amino acid sequence of SEQ ID NO:12.In certain embodiments, the anti-CD7 protein expression blockercomprises an amino acid sequence of SEQ ID NO:14, an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:15, and an amino acidsequence of SEQ ID NO:12. In other embodiments, the anti-CD7 proteinexpression blocker comprises an amino acid sequence having at least 90%sequence identity or at least 95% sequence identity to SEQ ID NO:14, anamino acid sequence having at least 90% sequence identity or at least95% sequence identity to SEQ ID NO:5, and an amino acid sequence of SEQID NO:12. In some instance, the anti-CD7 protein expression blocker alsocomprises a localization domain selected from any one sequence set forthin SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:13. In some cases, theanti-CD7 protein expression blocker also comprises a CD8α signal peptidesuch as but not limited to the CD8α signal peptide set forth in SEQ IDNO:7. In other cases, the anti-CD7 protein expression blocker alsocomprises a CD8α hinge and transmembrane domain such as but not limitedto the CD8α hinge and transmembrane domain set forth in SEQ ID NO:10.

In some embodiments, the anti-CD7 protein expression blocker comprisesan amino acid sequence of SEQ ID NO:16, an amino acid sequence of SEQ IDNO:17, and a VH-VL linker. The VH-VL linker can be a (GGGGS)_(n) linkerwhere n can range from 1 to 5, e.g., 1, 2, 3, 4, 5, or 6 (SEQ ID NO:29).In one embodiment, the anti-CD7 protein expression blocker comprises anamino acid sequence of SEQ ID NO:16, an amino acid sequence of SEQ IDNO:17, and an amino acid sequence of SEQ ID NO:12. In some embodiments,the anti-CD7 protein expression blocker comprises an amino acid sequencehaving at least 90% sequence identity or at least 95% sequence identityto SEQ ID NO:16, the amino acid sequence of SEQ ID NO:17, and the aminoacid sequence of SEQ ID NO:12. In certain embodiments, the anti-CD7protein expression blocker comprises an amino acid sequence of SEQ IDNO:16, an amino acid sequence having at least 90% sequence identity orat least 95% sequence identity to SEQ ID NO:17, and an amino acidsequence of SEQ ID NO:12. In other embodiments, the anti-CD7 proteinexpression blocker comprises an amino acid sequence having at least 90%sequence identity or at least 95% sequence identity to SEQ ID NO:16, anamino acid sequence having at least 90% sequence identity or at least95% sequence identity to SEQ ID NO:17, and an amino acid sequence of SEQID NO:12. In some instance, the anti-CD7 protein expression blocker alsocomprises a localization domain selected from any one sequence set forthin SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:13. In some cases, theanti-CD7 protein expression blocker also comprises a CD8α signal peptidesuch as but not limited to the CD8α signal peptide set forth in SEQ IDNO:7. In other cases, the anti-CD7 protein expression blocker alsocomprises a CD8α hinge and transmembrane domain such as but not limitedto the CD8α hinge and transmembrane domain set forth in SEQ ID NO:10.

In some embodiments, the nucleic acid sequence encoding an anti-CD7 PEBLcomprises one or more nucleic acid sequences set forth in Table 4. Insome embodiments, the VH domain of the anti-CD7 scFv of the PEBLcomprises the nucleotide sequence of SEQ ID NO:23 and the VL domain ofthe anti-CD7 scFv of the PEBL comprises the nucleotide sequence of SEQID NO:24. In certain embodiments, the VH domain of the anti-CD7 scFv ofthe PEBL comprises the nucleotide sequence having at least 90% sequenceidentity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity) to SEQ ID NO:23 and the VL domain of theanti-CD7 scFv of the PEBL comprises the nucleotide sequence having atleast 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more sequence identity) to SEQ ID NO:24.

TABLE 4 Nucleic acid sequence information for selectcomponents of an anti-CD7 PEBL based on TH69 Component SequenceCD8α signal ATGGCTCTGCCTGTGACCGCAC peptide TGCTGCTGCCCCTGGCTCTGCTGCTGCACGCCGCAAGACCT  (SEQ ID NO: 30) Anti-CD7 scFvGCCGCATACAAGGATATTCAGA VL (TH69) TGACTCAGACCACAAGCTCCCTGAGCGCCTCCCTGGGAGACCGA GTGACAATCTCTTGCAGTGCAT CACAGGGAATTAGCAACTACCTGAATTGGTATCAGCAGAAGCCA GATGGCACTGTGAAACTGCTGA TCTACTATACCTCTAGTCTGCACAGTGGGGTCCCCTCACGATTC AGCGGATCCGGCTCTGGGACAG ACTACAGCCTGACTATCTCCAACCTGGAGCCCGAAGATATTGCC ACCTACTATTGCCAGCAGTACT CCAAGCTGCCTTATACCTTTGGCGGGGGAACAAAGCTGGAGATT AAAAGG (SEQ ID NO: 24) Anti-CD7 scFvGAGGTGCAGCTGGTCGAATCTG VH (TH69) GAGGAGGACTGGTGAAGCCAGGAGGATCTCTGAAACTGAGTTGT GCCGCTTCAGGCCTGACCTTCT CAAGCTACGCCATGAGCTGGGTGCGACAGACACCTGAGAAGCGG CTGGAATGGGTCGCTAGCATCT CCTCTGGCGGGTTCACATACTATCCAGACTCCGTGAAAGGCAGA TTTACTATCTCTCGGGATAACG CAAGAAATATTCTGTACCTGCAGATGAGTTCACTGAGGAGCGAG GACACCGCAATGTACTATTGTG CCAGGGACGAAGTGCGCGGCTATCTGGATGTCTGGGGAGCTGGC ACTACCGTCACCGTCTCCAGC  (SEQ ID NO: 25)VH-VL Linker GGAGGAGGAGGAAGCGGAGGAG GAGGATCCGGAGGCGGGGGATCTGGAGGAGGAGGAAGT  (SEQ ID NO: 31) ER localization GAGCAGAAACTGATTAGCGAAGdomain KDEL AGGACCTGAAAGATGAACTG tethered to (SEQ ID NO: 32) scFv withmyc (“myc KDEL”)

In some embodiments, the nucleic acid sequence encoding the localizationdomain of the anti-CD7 protein expression blocker comprises a sequenceselected from SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, or a codonoptimized variant thereof.

In certain aspects of the present invention, the protein expressionblocker can bind to a molecule that is expressed on the surface of acell including, but not limited to members of the CD1 family ofglycoproteins, CD2, CD3ζ, CD4, CD5, CD7, CD8, CD25, CD28, CD30, CD38,CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, and CD137.

In some aspects of the present invention, expression of a member of theCD1 family of glycoproteins, CD2, CD3ζ, CD4, CD5, CD7, CD8, CD25, CD28,CD30, CD38, CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, orCD137 can be downregulated using a gene editing method, such as, but notlimited to, a gene editing technology that employs meganucleases, TALEN,CRISPR/Cas9, or zinc finger nucleases. For example, in some embodiments,CD7 expression is knocked out using genome editing by Cas9/CRISPR. Inother embodiments, CDS expression is knocked out using genome editing byCas9/CRISPR.

As noted above, downregulation of CD7 expression on the effector T cellscan be achieved according to a variety of other known methods including,for example, gene editing methods with meganucleases, TALEN,CRISPR/Cas9, and zinc finger nucleases. Thus, in certain embodiments,the engineered immune cell further comprises a modified CD7 gene, whichmodification renders the CD7 gene or protein non-functional. By way ofexample, the engineered immune cell of the present invention furthercomprises a modified (e.g., non-functional) CD7 gene (modified using,e.g., meganucleases, TALEN, CRISPR/Cas9, or zinc finger nucleases) thatprevents or reduces expression of CD7, and/or otherwise impairs (e.g.,structurally) the CD7 protein from being recognized by an anti-CD7 CAR.Methods of modifying gene expression using such methods are readilyavailable and well-known in the art.

Methods of inactivating a target gene in an immune cell usingCRISPR/Cas6 technology are described, for example, in US PatentPublication Nos. 2016/0272999, 2017/0204372, and 2017/0119820.

The CRISPR/Cas system is a system for inducing targeted geneticalterations (genome modifications). Target recognition by the Cas9protein requires a “seed” sequence within the guide RNA (gRNA) and aconserved multinucleotide containing protospacer adjacent motif (PAM)sequence upstream of the gRNA-binding region. The CRISPR/Cas system canthereby be engineered to cleave substantially any DNA sequence byredesigning the gRNA in cell lines, primary cells, and engineered cells.The CRISPR/Cas system can simultaneously target multiple genomic loci byco-expressing a single Cas9 protein with two or more gRNAs, making thissystem uniquely suited for multiple gene editing or synergisticactivation of target genes. Examples of a CRISPR/Cas system used toinhibit gene expression are described in U.S. Publication No.2014/0068797 and U.S. Pat. Nos. 8,697,359 and 8,771,945. The systeminduces permanent gene disruption that utilizes the RNA-guided Cas9endonuclease to introduce DNA double stranded breaks which triggererror-prone repair pathways to result in frame shift mutations. In somecases, other endonucleases may also be used, including but not limitedto, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (alsoknown as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1,Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5,Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1,Csx15, Csf1, Csf2, Csf3, Csf4, T7, Fok1, other nucleases known in theart, homologs thereof, or modified versions thereof.

CRISPR/Cas gene disruption occurs when a gRNA sequence specific for atarget gene and a Cas endonuclease are introduced into a cell and form acomplex that enables the Cas endonuclease to introduce a double strandbreak at the target gene. In some instances, the CRISPR system comprisesone or more expression vectors comprising a nucleic acid sequenceencoding the Cas endonuclease and a guide nucleic acid sequence specificfor the target gene. The guide nucleic acid sequence is specific for agene and targets that gene for Cas endonuclease-induced double strandbreaks. The sequence of the guide nucleic acid sequence may be within aloci of the gene. In some embodiment, the guide nucleic acid sequence isat least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or morenucleotides in length. The guide nucleic acid sequence includes a RNAsequence, a DNA sequence, a combination thereof (a RNA-DNA combinationsequence), or a sequence with synthetic nucleotides, such as a peptidenucleic acid (PNA) or Locked Nucleic Acid (LNA). The guide nucleic acidsequence can be a single molecule or a double molecule. In oneembodiment, the guide nucleic acid sequence comprises a single guideRNA.

In some embodiments, the engineered immune cell of the present inventioncan be modified via the CRISPR/Cas system to inactivate the human CD7gene. Details of the genomic structure and sequence of the human CD7gene can be found, for example, in NCBI Gene database under GeneID No.924.

Commercially available kits, gRNA vectors and donor vectors, forknockout of specific target genes are available, for example, fromOrigene (Rockville, Md.), GenScript (Atlanta, Ga.), Applied BiologicalMaterials (ABM; Richmond, British Colombia), BioCat (Heidelberg,Germany) or others. For example, commercially available kits or kitcomponents for knockout of CD7 via CRISPR include, for example, thoseavailable as catalog numbers KN201231, KN201231G1, KN201231G2, andKN201231D, each available from OriGene, and those available as catalognumbers sc-4072847, sc-4072847-KO-2, sc-4072847-HDR-2, sc-4072847-NIC,sc-4072847HDR-2, and sc-4072847-NIC-2, each available from Santa CruzBiotechnology.

In some embodiments, the chimeric antigen receptor described herein canbe introduced into the human CD7 gene locus using the CRISPR/Cas system.

In certain embodiments, provided is an engineered immune cellcomprising: i) a nucleic acid that comprises a nucleotide sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprisesintracellular signaling domains of 4-1BB and CD3ζ, and an antibody thatspecifically binds Cluster of Differentiation 7 (CD7); and ii) a nucleicacid that comprises a nucleotide sequence encoding a target-bindingmolecule linked to a localizing domain, wherein the target-bindingmolecule is an antibody that binds CD7, and the localizing domaincomprises an endoplasmic reticulum retention sequence. In certainembodiments, the antibody that binds CD7 in the context of the CAR, aswell as in the context of the target-binding molecule comprises: a VHsequence set forth in SEQ ID NO: 1 and a VL sequence set forth in SEQ IDNO: 2; a VH sequence set forth in SEQ ID NO: 14 and a VL sequence setforth in SEQ ID NO: 15; or a VH sequence set forth in SEQ ID NO: 16 anda VL sequence set forth in SEQ ID NO: 17. As described herein, incertain embodiments, the antibody comprises a VH and a VL havingsequence that each comprise at least 90% sequence identity, at least 91%sequence identity, at least 92% sequence identity, at least 93% sequenceidentity, at least 94% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, at least 99% sequenceidentity, or 100% sequence identity to the VH and VL sequences set forthin SEQ ID NO: 1 and 2, respectively; SEQ ID NO: 14 and SEQ ID NO: 15,respectively; or SEQ ID NO: 16 and SEQ ID NO: 17, respectively. Incertain embodiments, the antibody that binds CD7 in the context of theCAR can be different from the antibody that binds CD7 in the context ofthe target-binding molecule (the protein expression blocker or PEBL), asdescribed herein. In certain embodiments, the intracellular signalingdomain of 4-1BB comprises the sequence set forth in SEQ ID NO: 3. Incertain embodiments, the intracellular signaling domain of CD3ζcomprises the sequence set forth in SEQ ID NO: 4.

In another aspect, also provided is a nucleic acid comprising anucleotide sequence encoding a CAR, wherein the CAR comprisesintracellular signaling domains of 4-1BB and CD3ζ, and an antibody thatbinds CD7, as described herein.

In certain embodiments, the antibody is a scFv. In certain embodiments,the scFv comprises a VH sequence set forth in SEQ ID NO: 1 and avariable light chain VL sequence set forth in SEQ ID NO: 2. In certainembodiments, the scFv comprises a VH sequence set forth in SEQ ID NO: 14and a variable light chain VL sequence set forth in SEQ ID NO: 15. Incertain embodiments, the scFv comprises a VH sequence set forth in SEQID NO: 16 and a variable light chain VL sequence set forth in SEQ ID NO:17. As described herein, in certain embodiments, the scFv comprises a VHand a VL having sequence that each comprise at least 90% sequenceidentity, at least 91% sequence identity, at least 92% sequenceidentity, at least 93% sequence identity, at least 94% sequenceidentity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, at least 99% sequence identity, or 100% sequence identity tothe VH and VL sequences set forth in SEQ ID NO: 1 and 2, respectively;SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or SEQ ID NO: 16 and SEQID NO: 17, respectively. In certain embodiments, the CAR furthercomprises a hinge and transmembrane sequence.

In certain embodiments, an isolated nucleic acid of the presentinvention comprises a nucleotide sequence that encodes a CAR accordingto Table 5. In some embodiments, the nucleic acid comprises a nucleotidesequence that encodes a component of the CAR according to Table 5.

TABLE 5 Amino acid sequence information forselect components of an anti-CD7 CAR Component Amino Acid SequenceAnti-CD7 VH EVQLVESGGGLVKPGGSLKLSCAASG (TH69) LTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNIL YLQMSSLRSEDTAMYYCARDEVRGYL DVWGAGTTVTVSS (SEQ ID NO: 1) Anti-CD7 VL AAYKDIQMTQTTSSLSASLGDRVTIS (TH69)CSASQGISNYLNWYQQKPDGTVKLLI YYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGG GTKLEIKR (SEQ ID NO: 2) IntracellularKRGRKKLLYIFKQPFMRPVQTTQEED signaling GCSCRFPEEEEGGCEL  domain of 4-1BB(SEQ ID NO: 3) Intracellular RVKFSRSADAPAYQQGQNQLYNELNL signalingGRREEYDVLDKRRGRDPEMGGKPRRK domain CD3ζ NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO: 4) Hinge andTTTPAPRPPTPAPTIASQPLSLRPEA transmembrane CRPAAGGAVHTRGLDFACDIYIWAPLdomain AGTCGVLLLSLVITLY  of CD8α (SEQ ID NO: 10)

In some embodiments, the anti-CD7 CAR comprises an amino acid sequenceof SEQ ID NO:1, an amino acid sequence of SEQ ID NO:2, a 4-1BBintracellular signaling domain, a CD3ζ intracellular signaling domain,and a CD8 hinge and transmembrane domain. In some embodiments, theanti-CD7 CAR also includes a VH-VL linker such as but not limited to a(GGGGS)_(n) linker where n can range from 1 to 6, e.g., 1, 2, 3, 4, 5,or 6.

In one embodiment, the anti-CD7 protein expression blocker comprises anamino acid sequence of SEQ ID NO:1, an amino acid sequence of SEQ IDNO:2, an amino acid sequence of SEQ ID NO:3, an amino acid sequence ofSEQ ID NO:4, and an amino acid sequence of SEQ ID NO:10. In someembodiments, the anti-CD7 protein expression blocker comprises an aminoacid sequence having at least 90% sequence identity or at least 95%sequence identity to SEQ ID NO:1, an amino acid sequence having at least90% sequence identity or at least 95% sequence identity to SEQ ID NO:2,an amino acid sequence of SEQ ID NO:3, an amino acid sequence of SEQ IDNO:4, and an amino acid sequence of SEQ ID NO:10. In some embodiments,the anti-CD7 protein expression blocker comprises an amino acid sequencehaving at least 90% sequence identity or at least 95% sequence identityto SEQ ID NO:1, an amino acid sequence having at least 90% sequenceidentity or at least 95% sequence identity to SEQ ID NO:2, an amino acidsequence having at least 90% sequence identity or at least 95% sequenceidentity to SEQ ID NO:3, an amino acid sequence of SEQ ID NO:4, and anamino acid sequence of SEQ ID NO:10. In some embodiments, the anti-CD7protein expression blocker comprises an amino acid sequence having atleast 90% sequence identity or at least 95% sequence identity to SEQ IDNO:1, an amino acid sequence having at least 90% sequence identity or atleast 95% sequence identity to SEQ ID NO:2, an amino acid sequence ofSEQ ID NO:3, an amino acid sequence having at least 90% sequenceidentity or at least 95% sequence identity to SEQ ID NO:4, and an aminoacid sequence of SEQ ID NO:10. In some embodiments, the anti-CD7 proteinexpression blocker comprises an amino acid sequence having at least 90%sequence identity or at least 95% sequence identity to SEQ ID NO:1, anamino acid sequence having at least 90% sequence identity or at least95% sequence identity to SEQ ID NO:2, an amino acid sequence of SEQ IDNO:3, an amino acid sequence of SEQ ID NO:4, and an amino acid sequencehaving at least 90% sequence identity or at least 95% sequence identityto SEQ ID NO:10. In some embodiments, the anti-CD7 protein expressionblocker comprises an amino acid sequence having at least 90% sequenceidentity or at least 95% sequence identity to SEQ ID NO:1, an amino acidsequence having at least 90% sequence identity or at least 95% sequenceidentity to SEQ ID NO:2, an amino acid sequence having at least 90%sequence identity or at least 95% sequence identity to SEQ ID NO:3, anamino acid sequence having at least 90% sequence identity or at least95% sequence identity to SEQ ID NO:4, and an amino acid sequence havingat least 90% sequence identity or at least 95% sequence identity to SEQID NO:10.

In certain embodiments, an isolated nucleic acid of the presentinvention comprises one or more nucleotide sequences of Table 6. In someembodiments, the nucleic acid comprises a nucleotide sequence of acomponent of the CAR as set forth in Table 6.

TABLE 6 Amino acid sequence information forselect components of an anti-CD7 CAR Component Nucleic Acid SequenceAnti-CD7 VH GAGGTGCAGCTGGTCGAATCTGGAGG (TH69) AGGACTGGTGAAGCCAGGAGGATCTCTGAAACTGAGTTGTGCCGCTTCAGGC CTGACCTTCTCAAGCTACGCCATGAGCTGGGTGCGACAGACACCTGAGAAGC GGCTGGAATGGGTCGCTAGCATCTCCTCTGGCGGGTTCACATACTATCCAGA CTCCGTGAAAGGCAGATTTACTATCTCTCGGGATAACGCAAGAAATATTCTG TACCTGCAGATGAGTTCACTGAGGAGCGAGGACACCGCAATGTACTATTGTG CCAGGGACGAAGTGCGCGGCTATCTGGATGTCTGGGGAGCTGGCACTACCGT CACCGTCTCCAGC (SEQ ID NO: 23) Anti-CD7 VLGCCGCATACAAGGATATTCAGATGAC (TH69) TCAGACCACAAGCTCCCTGAGCGCCTCCCTGGGAGACCGAGTGACAATCTCT TGCAGTGCATCACAGGGAATTAGCAACTACCTGAATTGGTATCAGCAGAAGC CAGATGGCACTGTGAAACTGCTGATCTACTATACCTCTAGTCTGCACAGTGG GGTCCCCTCACGATTCAGCGGATCCGGCTCTGGGACAGACTACAGCCTGACT ATCTCCAACCTGGAGCCCGAAGATATTGCCACCTACTATTGCCAGCAGTACT CCAAGCTGCCTTATACCTTTGGCGGGGGAACAAAGCTGGAGATTAAAAGG  (SEQ ID NO: 24) IntracellularAAGCGGGGGCGCAAAAAACTGCTGTA signaling domain TATCTTTAAGCAGCCTTTCATGAGACof4-1BB CAGTGCAGACAACCCAGGAGGAAGAT GGGTGCTCATGCCGGTTTCCCGAGGAGGAGGAAGGCGGCTGCGAGCTG  (SEQ ID NO: 35) IntracellularAGGGTGAAGTTTTCCCGCTCAGCAGA signaling domain TGCTCCTGCCTACCAGCAGGGCCAGAof CD3ζ ACCAGCTGTATAATGAGCTGAACCTG GGCAGACGCGAAGAGTATGATGTGCTGGACAAAAGGCGGGGAAGAGACCCCG AAATGGGAGGGAAGCCAAGGCGGAAAAACCCCCAGGAGGGCCTGTACAATGA GCTGCAGAAGGACAAAATGGCAGAGGCTTACAGTGAGATTGGGATGAAGGGA GAGAGACGGAGGGGAAAAGGGCACGATGGCCTGTACCAGGGGCTGAGCACAG CAACCAAAGATACTTATGACGCACTGCACATGCAGGCACTGCCACCCAGA  (SEQ ID NO: 36) Hinge andACCACTACACCTGCACCAAGGCCTCC transmembrane CACACCCGCTCCCACTATCGCTTCCCdomain of CD8α AGCCACTGTCCCTGAGGCCCGAGGCC TGCAGGCCAGCAGCTGGCGGAGCCGTGCATACTAGGGGGCTGGACTTCGCTT GCGACATCTACATCTGGGCCCCACTGGCAGGGACATGCGGAGTCCTGCTGCT GTCCCTGGTCATCACACTGTAC (SEQ ID NO: 37)

In certain embodiments, a nucleic acid further comprises a nucleotidesequence that encodes a target-binding molecule linked to a localizingdomain, as described herein. In certain embodiments, the target-bindingmolecule is an antibody that binds CD7. In certain embodiments, theantibody is a scFv. In some embodiments, the scFv comprises a VHsequence having at least 90% sequence identity (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to thesequence of SEQ ID NO: 1 and a VL sequence having at least 90% sequenceidentity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity) to the sequence of SEQ ID NO: 2. In certainembodiments, the scFv comprises a VH sequence set forth in SEQ ID NO: 1and a VL sequence set forth in SEQ ID NO: 2. In some embodiments, the VHdomain of the anti-CD7 scFv comprises the nucleotide sequence of SEQ IDNO:23 and the VL domain of the anti-CD7 scFv comprises the nucleotidesequence of SEQ ID NO:24.

In other aspects, also provided is a method of treating cancer in asubject in need thereof, comprising administering a therapeutic amountof an engineered immune cell having any of the embodiments describedherein to the subject, thereby treating cancer in a subject in needthereof.

In certain embodiments, the method comprises administering a therapeuticamount of an engineered immune cell comprising a nucleic acid thatcomprises a nucleotide sequence encoding a CAR, wherein the CARcomprises intracellular signaling domains of 4-1BB and CD3ζ, and anantibody that binds CD7, as described herein.

In certain embodiments, the method comprises administering a therapeuticamount of an engineered immune cell that further comprises a nucleicacid having a nucleotide sequence encoding a target-binding moleculelinked to a localizing domain, as described herein (e.g., an anti-CD7protein expression blocker).

In certain embodiments, the cancer is a T cell malignancy, e.g., T cellleukemia or T cell lymphoma, such a T-cell acute lymphoblastic leukemia,T-cell prolymphocytic leukemia, T-cell large granular lymphocyticleukemia, enteropathy-associated T-cell lymphoma, hepatosplenic T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosisfungoides, Sézary syndrome, primary cutaneous gamma-delta T-celllymphoma, peripheral T-cell lymphoma not otherwise specified,angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma. Incertain embodiments, the T cell malignancy is early T-cell progenitoracute lymphoblastic leukemia (ETP-ALL).

As used herein, the terms “treat,” “treating,” or “treatment,” refer tocounteracting a medical condition (e.g., a condition related to a T cellmalignancy) to the extent that the medical condition is improvedaccording to a clinically-acceptable standard.

As used herein, “subject” refers to a mammal (e.g., human, non-humanprimate, cow, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat,mouse). In certain embodiments, the subject is a human. A “subject inneed thereof” refers to a subject (e.g., patient) who has, or is at riskfor developing, a disease or condition that can be treated (e.g.,improved, ameliorated, prevented) by inducing T cells to exert specificcytotoxicity against malignant T cells.

As defined herein, a “therapeutic amount” refers to an amount that, whenadministered to a subject, is sufficient to achieve a desiredtherapeutic effect (treats a condition related to a T cell malignancy)in the subject under the conditions of administration. An effectiveamount of the agent to be administered can be determined by a clinicianof ordinary skill using the guidance provided herein and other methodsknown in the art, and is dependent on several factors including, forexample, the particular agent chosen, the subject's age, sensitivity,tolerance to drugs and overall well-being.

In some embodiments, the engineered immune cell is autologous to thesubject in need of treatment, e.g., cancer treatment. In otherembodiments, the engineered immune cell is allogenic to the subject inneed of treatment.

In certain embodiments, the engineered immune cell is administered intothe subject by intravenous infusion, intra-arterial infusion, directinjection into tumor and/or perfusion of tumor bed after surgery,implantation at a tumor site in an artificial scaffold, intrathecaladministration, and intraocular administration.

In certain embodiments, the engineered immune cell is administered byinfusion into the subject. Methods of infusing immune cells (e.g.,allogeneic or autologous immune cells) are known in the art. Asufficient number of cells are administered to the recipient in order toameliorate the symptoms of the disease. Typically, dosages of 10⁷ to10¹⁰ cells are infused in a single setting, e.g., dosages of 10⁹ cells.Infusions are administered either as a single 10⁹ cell dose or dividedinto several 10⁹ cell dosages. The frequency of infusions can be daily,every 2 to 30 days or even longer intervals if desired or indicated. Thequantity of infusions is generally at least 1 infusion per subject andpreferably at least 3 infusions, as tolerated, or until the diseasesymptoms have been ameliorated. The cells can be infused intravenouslyat a rate of 50-250 ml/hr. Other suitable modes of administrationinclude intra-arterial infusion, intraperitoneal infusion, directinjection into tumor and/or perfusion of tumor bed after surgery,implantation at the tumor site in an artificial scaffold, intrathecaladministration. Methods of adapting the present invention to such modesof delivery are readily available to one skilled in the art.

In certain embodiments, the method of treating cancer according to thepresent invention is combined with at least one other known cancertherapy, e.g., radiotherapy, chemotherapy, or other immunotherapy.

In other aspects, also provided is use of an engineered immune cellhaving any of the embodiments described herein for treating cancer,comprising administering a therapeutic amount of the engineered immunecell to a subject in need thereof. In certain embodiments, the cancer isa T cell malignancy. In certain embodiments, the T cell malignancy isearly T-cell progenitor acute lymphoblastic leukemia (ETP-ALL).

In certain embodiments, the engineered immune cell is administered intothe subject by intravenous infusion, intra-arterial infusion,intraperitoneal infusion, direct injection into tumor and/or perfusionof tumor bed after surgery, implantation at a tumor site in anartificial scaffold, and intrathecal administration.

In another aspect, also provided is a method for producing theengineered immune cell having any of the embodiments described herein,the method comprising introducing into an immune cell a nucleic acidthat comprises a nucleotide sequence encoding a CAR, wherein the CARcomprises intracellular signaling domains of 4-1BB and CD3ζ, and anantibody that binds CD7.

In certain embodiments, the method further comprises introducing intothe immune cell a nucleic acid that comprises a nucleotide sequenceencoding a target-binding molecule linked to a localizing domain (e.g.,anti-CD7 protein expression blocker or anti-CD7 PEBL). In certainembodiments, the nucleotide sequence encoding CAR and the nucleotidesequence encoding the anti-CD7 PEBL are introduced on a single plasmid.

In various aspects, also provided is a kit for producing an engineeredimmune cell described herein. The present kit can be used to produce,e.g., allogeneic or autologous T cells having anti-CD7 CAR-mediatedcytotoxic activity. In some embodiments, the kit is useful for producingallogeneic effector T cells having anti-CD7 CAR-mediated cytotoxicactivity. In certain embodiments, the kit is useful for producingautologous effector T cells having anti-CD7 CAR-mediated cytotoxicactivity.

Accordingly, provided herein is a kit comprising a nucleic acidcomprising a nucleotide sequence encoding a CAR, wherein the CARcomprises intracellular signaling domains of 4-1BB and CD3ζ, and anantibody that binds CD7. The nucleotide sequence encoding the anti-CD7CAR can be designed according to any of the embodiments describedherein. In certain embodiments, the nucleotide sequence encodes theanti-CD7 CAR according to the schematic in FIG. 1A (“anti-CD7-41BB-CD3ζconstruct”).

In certain embodiments, the kit further comprises a nucleic acid havinga nucleotide sequence that encodes a target-binding molecule linked to alocalizing domain, as described herein (e.g., anti-CD7 PEBL moleculesdescribed herein). The nucleotide sequence encoding the target-bindingmolecule linked to a localizing domain can be designed according to anyof the embodiments described herein.

In certain embodiments, the nucleotide sequence encoding the anti-CD7CAR and/or the nucleotide sequence encoding the anti-CD7 PEBL furthercomprise sequences (e.g., plasmid or vector sequences) that allow, e.g.,cloning and/or expression. For example, the nucleotide sequence can beprovided as part of a plasmid for ease of cloning into other plasmidsand/or vectors (expression vectors or viral expression vectors) for,e.g., transfection, transduction, or electroporation into a cell (e.g.,an immune cell). In certain embodiments, the nucleotide sequenceencoding the anti-CD7 CAR and the nucleotide sequence encoding theanti-CD7 PEBL are provided on a single plasmid or vector (e.g., a singleconstruct comprising an anti-CD7 CAR and an anti-CD7 PEBL). In certainembodiments, the nucleotide sequences are provided on separate plasmidsor vectors (expression vectors or viral expression vectors).

Typically, the kits are compartmentalized for ease of use and caninclude one or more containers with reagents. In certain embodiments,all of the kit components are packaged together. Alternatively, one ormore individual components of the kit can be provided in a separatepackage from the other kits components. The kits can also includeinstructions for using the kit components.

In some embodiments, provided herein is an engineered immune cellcomprising a nucleic acid that comprises a nucleotide sequence encodinga chimeric antigen receptor (CAR), wherein the CAR comprisesintracellular signaling domains of 4-1BB and CD3ζ, and an antibody thatbinds Cluster of Differentiation 7 (CD7). In certain embodiments, theantibody is a single chain variable fragment (scFv). In some instances,the scFv comprises a heavy chain variable domain (VH) sequence set forthin SEQ ID NO: 1 and a light chain variable domain (VL) sequence setforth in SEQ ID NO: 2.

In some embodiments, the CAR further comprises a hinge and transmembranesequence, such as but not limited to a hinge and transmembrane domaincomprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the engineered immune cell is an engineered T cell,an engineered natural killer (NK) cell, an engineered NK/T cell, anengineered monocyte, an engineered macrophage, or an engineereddendritic cell.

In some embodiments, the engineered immune cell further comprising anucleic acid that comprises a nucleotide sequence encoding atarget-binding molecule linked to a localizing domain. In certainembodiments, the target-binding molecule is an antibody that binds CD7.In certain embodiments, the antibody is an scFv. In some embodiments,the scFv comprises a VH sequence set forth in SEQ ID NO: 1 and a VLsequence set forth in SEQ ID NO: 2. In some embodiments, the localizingdomain comprises an endoplasmic reticulum (ER) or Golgi retentionsequence; a proteosome localizing sequence; a transmembrane domainsequence derived from CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16,OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B.

In some embodiments, provided herein is an engineered immune cellcomprising: (i) a nucleic acid that comprises a nucleotide sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprisesintracellular signaling domains of 4-1BB and CD3ζ, and an antibody thatbinds Cluster of Differentiation 7 (CD7); and (ii) a nucleic acid thatcomprises a nucleotide sequence encoding a target-binding moleculelinked to a localizing domain, wherein the target-binding molecule is anantibody that binds CD7, and the localizing domain comprises anendoplasmic reticulum retention sequence, and wherein the antibody thatbinds CD7 comprises a variable heavy chain (VH) sequence set forth inSEQ ID NO: 1 and a variable light chain (VL) sequence set forth in SEQID NO: 2. In some embodiments, the intracellular signaling domain of4-1BB comprises the sequence set forth in SEQ ID NO: 3 and theintracellular signaling domain of CD3ζ comprises the sequence set forthin SEQ ID NO: 4.

In some embodiments, provided herein is an method of treating cancer ina subject in need thereof, comprising administering a therapeutic amountof the engineered immune cell described herein to the subject, therebytreating cancer in a subject in need thereof. In some embodiments, thecancer is a T cell malignancy. In certain embodiments, the T cellmalignancy is early T-cell progenitor acute lymphoblastic leukemia(ETP-ALL). In certain embodiments, the engineered immune cell isadministered into the subject by intravenous infusion, intra-arterialinfusion, intraperitoneal infusion, direct injection into tumor and/orperfusion of tumor bed after surgery, implantation at a tumor site in anartificial scaffold, intrathecal administration.

In some embodiments, provided herein is a nucleic acid comprising anucleotide sequence encoding a chimeric antigen receptor (CAR), whereinthe CAR comprises intracellular signaling domains of 4-1BB and CD3ζ, andan antibody that binds Cluster of Differentiation 7 (CD7).

In other embodiments, provided herein is the of the engineered immunecell described herein for treating cancer comprising administering atherapeutic amount of the engineered immune cell to a subject in needthereof. In some embodiments, the cancer is a T cell malignancy. Incertain embodiments, the T cell malignancy is early T-cell progenitoracute lymphoblastic leukemia (ETP-ALL). In certain embodiments, theengineered immune cell is administered into the subject by intravenousinfusion, intra-arterial infusion, intraperitoneal infusion, directinjection into tumor and/or perfusion of tumor bed after surgery,implantation at a tumor site in an artificial scaffold, intrathecaladministration.

In some embodiments, provided herein is a method for producing theengineered immune cell described herein. The method can include:introducing into an immune cell a nucleic acid that comprises anucleotide sequence encoding a CAR, wherein the CAR comprisesintracellular signaling domains of 4-1BB and CD3ζ, and an antibody thatbinds CD7, thereby producing an engineered immune cell. The method canfurther comprise introducing into the immune cell a nucleic acid thatcomprises a nucleotide sequence encoding a target-binding moleculelinked to a localizing domain.

The present invention provides a chimeric antigen receptor (CAR)directed against CD7. As demonstrated herein, the expression of theanti-CD7 CAR in immune cells such as effector T cells, induces the Tcell to exert specific cytotoxicity against T cell malignancies. Thiscytotoxic effect was shown to be enhanced when expression of CD7 on theeffector T cells was downregulated using an antibody-based molecule (aprotein expression blocker or PEBL) that targeted the CD7 fordownregulation. Thus, the present invention provides animmunotherapeutic method for treating cancers, e.g., T-cellmalignancies.

In some aspects, the present invention provides an engineered immunecell comprising a nucleic acid that comprises a nucleotide sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprisesintracellular signaling domains of 4-1BB and CD3ζ, and an antibody thatbinds Cluster of Differentiation 7 (CD7). In some embodiments, theengineered immune cell outlined herein also includes a nucleic acid thatcomprises a nucleotide sequence encoding a target-binding moleculelinked to a localizing domain (e.g., a protein expression blocker orPEBL). Outlined herein is also a method and kit for producing such anengineered immune cell.

In some aspects, the present invention provides an engineered immunecell (e.g., T cell, natural killer (NK) cell, NK/T cell, monocyte,macrophage, or dendritic cell) comprising (i) a nucleic acid thatcomprises a nucleotide sequence encoding a chimeric antigen receptor(CAR), wherein the CAR comprises intracellular signaling domains of4-1BB and CD3ζ, and an antibody that specifically binds CD7; and (ii) anucleic acid that comprises a nucleotide sequence encoding atarget-binding molecule linked to a localizing domain, wherein thetarget-binding molecule is an antibody that binds CD7, and thelocalizing domain comprises an endoplasmic reticulum retention sequence,and wherein the antibody that binds CD7 comprises a variable heavy chain(VH) sequence set forth in SEQ ID NO: 1 and a variable light chain (VL)sequence set forth in SEQ ID NO: 2.

In other aspects, the present invention provides a method of treatingcancer (e.g., a T cell malignancy) in a subject in need thereof. Themethod includes administering a therapeutic amount of any of theengineered immune cells described herein to the subject, therebytreating cancer in a subject in need thereof. The disclosure also setsforth the use of any of the engineered immune cells outlined herein fortreating cancer.

In other aspects, the present invention provides a nucleic acidcomprising a nucleotide sequence encoding a chimeric antigen receptor(CAR), wherein the CAR comprises intracellular signaling domains of4-1BB and CD3ζ, and an antibody that specifically binds CD7.

EXAMPLES Example 1: Blockade of Cd7 Expression in T Cells for EffectiveChimeric Antigen-Receptor Targeting of T-Cell Malignancies

This example illustrates blockade of CD7 expression with a novel method,combined with a second-generation CAR, resulted in highly potentanti-CD7 CAR-T cells. This practical strategy provides a new treatmentoption for patients with high-risk T-cell malignancies, includingETP-ALL.

Abstract

Effective immunotherapies for T-cell malignancies are lacking. A novelapproach based on chimeric antigen receptor (CAR)-redirected Tlymphocytes was devised. CD7 was selected as a target because of itsconsistent expression in T-cell acute lymphoblastic leukemia (T-ALL),including the most aggressive subtype, early T-cell precursor (ETP)-ALL.In 49 diagnostic T-ALL samples (including 14 ETP-ALL), median CD7expression was >99%; CD7 expression remained high at relapse (n=14), andduring chemotherapy (n=54). CD7 was targeted with a second-generationCAR (anti-CD7-41BB-CD3ζ) but CAR expression in T lymphocytes causedfratricide, owing to CD7 present in the T cells themselves. Todownregulate CD7 and control fratricide, a new method (ProteinExpression Blocker, PEBL), based on an anti-CD7 single chain variablefragment coupled with an intracellular retention domain was applied.Transduction of anti-CD7 PEBL resulted in virtually instantaneousabrogation of surface CD7 expression in all transduced T cells;2.0%±1.7% were CD7+ versus 98.1%±1.5% of mock-transduced T cells (n=5;P<0.0001). PEBL expression did not impair T-cell proliferation, IFNγ andTNFα secretion, or cytotoxicity, and eliminated CAR-mediated fratricide.PEBL-CAR-T cells were highly cytotoxic against CD7+ leukemic cells invitro, and were consistently more potent than CD7+ T cells spared byfratricide. They also showed strong anti-leukemic activity in cell line-and patient-derived T-ALL xenografts. The strategy described here fitswell with existing clinical-grade cell manufacturing processes, and canbe rapidly implemented for the treatment of patients with high-riskT-cell malignancies.

INTRODUCTION

T lymphocytes can be induced to specifically recognize and kill tumorcells through the expression of chimeric antigen receptors (CARs).¹⁻⁵Central to the effective application of this technology is theidentification of a suitable target for the CAR. This must be highlyexpressed by tumor cells and should be absent in normal cells, or beexpressed only by normal cells whose temporary absence is clinicallymanageable.⁶ Thus, leukemias and lymphomas of B-cell origin can betargeted with CARs directed against CD19,^(5,7) or CD22,⁸ which arenormally expressed only by B lymphoid cells.^(9,10) Infusion ofautologous T cells expressing anti-CD19 CARs in patients with B-cellrefractory leukemia and lymphoma resulted in major clinicalresponses.¹¹⁻¹⁸ These exciting results have provided indisputableevidence of the power of this technology, and suggest the possibility ofwider applications in oncology.

The development of CAR-T cell therapies for T-cell malignancies haslagged far behind that of their B-cell counterparts. The need foreffective therapies in this area is particularly urgent because of thepoor prognosis associated with some T-cell leukemia and lymphomasubtypes. For example, children and adolescents with early T-cellprogenitor acute lymphoblastic leukemia (ETP-ALL) have the poorestresponse to initial therapy among all patients with ALL.¹⁹⁻²¹ Intensivechemotherapy and/or allogeneic hematopoietic stem cell transplant oftendo not prevent treatment-refractory relapse; for these patients, andthose with other high-risk features, such as adult age, there is adearth of treatment options.^(19,22-25)

A major obstacle to the development of effective CAR-T cells for T-cellmalignancies is that the surface marker profile of malignant T cells(which generally lack CD19 or CD22 expression) largely overlaps that ofactivated T lymphocytes.^(19,26) CAR directed against such targets arelikely to lead to the self-elimination of the CAR-T cells.^(27,28)Described herein is the development and application of a practicaltechnology for CAR-T cell therapy of ETP-ALL and other T-ALL cellsubtypes. First, a CAR directed against CD7 was made. As one recognizes,CD7 is a 40 kDa type I transmembrane glycoprotein that is a primarymarker for T-cell malignancies,²⁹⁻³² and is highly expressed in allcases of T-cell ALL, including ETP-ALL.¹⁹ Second, a method to rapidlyand effectively downregulate CD7 expression in T cells was developed.The method was selected as it averts the fratricide effect of CAR-T celltherapy, does not involve gene editing, and can be immediatelytranslated into clinical applications.

Materials and Methods

Cells and Culture Conditions

The leukemia cell lines Jurkat, CCRF-CEM, Loucy, MOLT4 and KG1a werefrom the American Type Culture Collection (ATCC; Rockville, Md.). TheB-lineage ALL cell line OP-1 was developed in our laboratory.³³ TheCCRF-CEM cells were transduced with a murine stem cell virus(MSCV)—internal ribosome entry site (IRES)—green fluorescent protein(GFP) retroviral vector (from the Vector Development and ProductionShared Resource of St. Jude Children's Research Hospital, Memphis,Tenn.) containing the firefly luciferase gene. The same vector was usedto transduce CCRF-CEM and Jurkat cells with the CD19 gene, which wascloned from the cDNA of the RS4;11 B-cell line (ATCC). Cell lines weremaintained in RPMI-1640 (ThermoFisher Scientific, Waltham, Mass.)supplemented with 10% fetal bovine serum (FBS) and 1%penicillin-streptomycin.

Peripheral blood samples were obtained from discarded anonymizedby-products of platelet donations from healthy adult donors at theNational University Hospital Blood Bank, Singapore. Bone marrowaspirates from patients with ALL were obtained for diagnosticimmunophenotyping, and monitoring of treatment response,^(19,26) bankedsurplus material was used in some experiments, with approval from theInstitutional Review Board, National University of Singapore.Mononucleated cells were separated by centrifugation on a Lymphoprepdensity step (Axis-Shield, Oslo, Norway) and washed twice in RPMI-1640.T cells were enriched with Dynabeads Human T-Activator CD3/CD28(ThermoFisher) and cultured in RPMI-1640, 10% FBS, 1%penicillin-streptomycin, and interleukin-2 (IL-2; 120 IU/mL; Proleukin,Novartis, Basel, Switzerland).

Gene Cloning and Retroviral Transduction

The single chain variable fragment (scFv) of the anti-CD7 monoclonalantibody TH69³⁴ was joined to the CD8α signal peptide, CD8α hinge andtransmembrane domain, and the intracellular domains of 4-1BB and CD3ζ ofan anti-CD19-41BB-CD3ζ CAR previously developed in our laboratory.⁵ Thesame scFv was also joined to the CD8α signal peptide and sequencesencoding endoplasmic reticulum (ER)/Golgi retention peptidesEQKLISEEDLKDEL (SEQ ID NO:8), (GGGGS)₄AEKDEL (SEQ ID NO:9), or CD8αhinge and transmembrane domain followed by localizing sequence (SEQ IDNO:13). These were subcloned into the MSCV vector, with or without GFPor mCherry.

Preparation of retroviral supernatant and transduction were performed aspreviously described.^(5,35) Briefly, pMSCV retroviralvector-conditioned medium was added to RetroNectin (Takara, Otsu,Japan)-coated polypropylene tubes; after centrifugation and removal ofthe supernatant, T cells were added to the tubes and left at 37° C. for12 hours; fresh viral supernatant was added on two other successivedays. T lymphocytes were maintained in RPMI-1640 with FBS, antibioticsand 200 IU/mL IL-2.

For transient CAR expression, anti-CD7 and anti-CD19 CAR constructs weresubcloned into EcoRI and XhoI sites of the pVAXI vector (ThermoFisherScientific), and transcribed into mRNA using T7 mScript (CellScript,Madison, Wis.).³⁶ For mRNA electroporation, cells were suspended inelectroporation buffer (Amaxa Cell Line Nucleofector Kit V; Lonza,Basel, Switzerland) containing 200 μg of CAR mRNA, and electroporatedwith an Amaxa Nucleofector 2b (Lonza) using program X-001. ^(36,37)Cells electroporated without mRNA were used as control.

Detection of CAR, PEBL and Surface Markers

CARs were detected with a biotin-conjugated goat anti-mouse F(ab′)₂antibody (Jackson ImmunoResearch, West Grove, Pa.) followed byallophycocyanin (APC)-conjugated streptavidin (Jackson ImmunoResearch).Phycoerythrin (PE)- or APC-conjugated anti-CD7 (M-T701), CD4 (RPA-T4),CD8 (RPA-T8), CD3ζ (SK7), and non-reactive isotype-matched antibodieswere from BD Biosciences (San Jose, Calif.); CD19 (LT19) was fromMiltenyi Biotech. Cell staining was analyzed using Accuri C6, Fortessaor LSRII flow cytometers (BD Biosciences), with Diva (BD Biosciences) orFlowJo software (FlowJo, Ashland, Oreg.).

Western blotting was performed as previously described.³⁵ Briefly, celllysates were extracted using CelLytic M cell lysis reagent(Sigma-Aldrich, Saint Louis, Mo.) prior to protein quantification withPierce BCA protein assay kit (ThermoFisher). Cell lysates were dilutedwith 4× Laemmli sample buffer (Bio-rad, Hercules, Calif.) and separatedon 10% polyacrylamide gel by electrophoresis under reducing ornon-reducing conditions. Blotted membranes were probed with mouseanti-human CD3ζ antibody (8D3; BD Biosciences), goat anti-mouse IgGhorseradish peroxidase-conjugated (R&D Systems, Minneapolis, Minn.), andClarity Western ECL substrate (Bio-Rad). Staining was visualised usingChemiDoc Touch Imager (Bio-Rad).

Cell Aggregation Assay, Cytotoxicity Assays and Cytokine Production

To measure cell-cell aggregation, Jurkat cells were co-cultured with theCD7+ or CD7− cells labeled with calcein red-orange AM (ThermoFisher) for30 minutes; cell doublets were counted by flow cytometry. In someexperiments, target cells were pre-incubated for 10 minutes beforeco-culture with a soluble anti-CD7 scFv, obtained from the supernatantof Jurkat or 293T cells transduced with a construct consisting of thescFv without transmembrane or signaling sequences.

To test cytotoxicity, target cells were labeled with calcein red-orangeAM and placed into a 96-well round bottom plate (Corning Costar,Corning, N.Y.). T cells were added at different effector: target (E:T)ratios with target cells and cultured for 4 hours at 37° C. and 5% CO2.Viable target cells were counted by flow cytometry.38 To measureexocytosis of lytic granules, anti-human CD107a-PE (H4A3; BDBiosciences) was added to the co-cultures. After 1 hour, monensin (BDGolgiStop) was added, and the cultures were continued for another 3hours before flow cytometric analysis.

To assess cell proliferation, T-cells were cultured alone or in presenceof MOLT-4 cells at 1:1 E:T in RPMI-1640 with FBS and 120 IU/mL IL-2 at37° C. and 5% CO₂. Target cells, irradiated or treated with Streck cellpreservative (Streck Laboratories, Omaha, Nebr.) to inhibitproliferation, were added to the cultures every 7 days. Viable GFP+ ormCherry+ T-cells were enumerated by flow cytometry. For IFNγ and TNFαproduction, target and effector cells at 1:1 E:T were plated as above.After 1 hour, brefeldin A (BD GolgiPlug) was added to the cultures,which continued for another 5 hours. Subsequently, intracellularstaining with anti-IFNγ-PE (clone 25723.11; BD Biosciences) oranti-TNFα-PE (6401.1111; BD Biosciences) was done prior to flowcytometric analysis.

Xenograft Models

CCRF-CEM cells transduced with luciferase were injected intravenously(i.v.) in NOD.Cg-Prkdc^(scid) IL2rg^(tm1wjl)/SzJ (NOD/scid IL2RGnull)mice (Jackson Laboratory, Bar Harbor, Me.) at 1×10⁶ cells per mouse.Three and/or seven days later, mice received T cells with downregulatedCD7 and anti-CD7 CAR expression at 2×10⁷ T cells per mouse. Other micereceived T cells transduced with GFP alone, or RPMI-1640 with 10% FBSinstead of T cells. All mice received 20,000 IU of IL-2intraperitoneally (i.p.) every 2 days. Tumor load was determined usingthe Xenogen IVIS-200 System (Caliper Life Sciences, Waltham, Mass.)after injecting aqueous D-luciferin potassium salt (Perkin Elmer,Waltham, Mass.) i.p. (2 mg per mouse). Luminescence was analyzed withthe Living Image 3.0 software. Mice were euthanized when luminescencereached 1×10¹⁰ photons per second, or earlier if physical signswarranting euthanasia appeared.

For the patient-derived xenograft (PDX) model, primary ETP-ALL cellswere injected i.v. in NOD/scid IL2RGnull and propagated for 7-8subsequent generations. ETP-ALL cells were then re-injected in NOD/scidIL2RGnull which were either treated with PEBL-CAR-T cells or leftuntreated. Peripheral blood and tissues were monitored for the presenceof ALL cells by flow cytometry. ^(19,26) After red blood cells lysiswith a lysing buffer (Sigma-Aldrich), cells were stained with anti-mouseCD45-PE-Cyanine 7 (30-F11, Biolegend), as well as anti-human CD45-APC-H7(2D1), CD7-PE (M-T701), CD3 APC (SK7), CD34-peridinin chlorophyllprotein (8G12) (all from BD Biosciences), and CD33-Brilliant Violet 421(WM53, Biolegend). Cells were analyzed with a Fortessa flow cytometer,using Diva and FlowJo software.

Results

Validation of CD7 as a Target for CAR-T Cell Therapy in Leukemia

In leukemic cells from diagnostic bone marrow samples obtained from 49patients with T-ALL (including 14 with ETP-ALL), median percent CD7expression was >99% (range, 79%->99%). In only 3 cases (6.1%), CD7 waslower than 99%: 98% in two, and 79% in one (FIG. 1A). High CD7expression was also observed in samples collected from 14 patients withrelapse T-ALL (FIG. 1A). Mean fluorescence intensity (MFI) of CD7 inleukemic cells at diagnosis or relapse consistently exceeded thatmeasured in residual normal T cells in the same samples. Median (range)MFI was 20,617 (4,105-66,674) in T-ALL cells versus 3,032 (1,301-9,582)in the normal T cells (n=19; P<0.0001) (FIG. 1B).

To determine whether chemotherapy affected CD7 expression, bone marrowsamples collected during therapy that contained minimal residual disease(MRD) were examined. In all 54 samples (from 21 patients), >99% ofresidual leukemic cells were CD7+ (FIG. 1A). In 18 patients, CD7 levelswere monitored during the course of the disease. As shown in FIG. 1C andFIG. 1D, CD7 remained high during therapy. These results validate CD7 asa target for CAR-T cell therapy in T-ALL.

Design and Expression of an Anti-CD7 CAR

To target CD7, an anti-CD7 CAR composed of the scFv of the anti-CD7antibody TH69 joined to the signaling domains of 4-1BB (CD137) and CD3ζvia the hinge and transmembrane domain of CD8α (FIG. 2A) was designed.Retroviral transduction of this construct in Jurkat cells resulted inhigh expression of anti-CD7 CAR (FIG. 2B), which appeared as monomer,dimer and oligomer by western blotting (FIG. 2C).

To confirm that the TH69 scFv could bind CD7, it was produced in solubleform and was tested on CD7+ MOLT-4 and CD7− OP-1 cells; MOLT-4 cellswere labelled while OP-1 were not (FIG. 8A). Further, staining with ananti-CD7 monoclonal antibody was significantly reduced when MOLT-4 cellswere pre-incubated with the anti-CD7 scFv supernatant; CD7 MFI (±SD)went from 31,730±1,144 to 5,987±241 (n=3). Jurkat cells expressinganti-CD7 CAR formed aggregates with CD7+ MOLT-4 cells, whereas thosetransduced with GFP only, or with an anti-CD19 CAR, did not; conversely,the anti-CD19 CAR induced cell aggregation with CD19+ OP-1 cells whilethe anti-CD7 CAR did not (FIG. 8B). Pre-incubation of MOLT-4 or CCRF-CEMwith the soluble anti-CD7 scFv prevented the formation of aggregates(FIG. 8C).

To determine whether the anti-CD7 CAR was functional, levels of theactivation markers CD25 and CD69 were measured in Jurkat cells after24-hour co-culture with MOLT4. There was a clear upregulation of bothactivation markers in cells expressing the anti-CD7 CAR (FIGS. 2D and2E). In sum, the anti-CD7-41BB-CD3ζ CAR can bind to its cognate antigen,and transduces activation signals upon ligation.

Expression of Anti-CD7 CAR in T Cells Causes Fratricide

To determine the effects of anti-CD7-41BB-CD3ζ CAR in peripheral blood Tlymphocytes, two different methods were used to express it: retroviraltransduction (FIG. 9A) and mRNA electroporation. However, it markedlyreduced T-cell viability. Mean (±SD) T-cell recovery 24 hours after mRNAelectroporation was 39.8%±13.0 (n=7) of the recovery afterelectroporation without mRNA (FIG. 3A); if the CAR was introduced byviral transduction, cell recovery was 25.1%±16.2% (n=10) of that ofmock-transduced T cells (FIG. 3B); overall, CAR expression reduced cellrecovery to 31.1%±16.3% (n=17) after 24 hours. Prolonging cell culturefurther increased the difference in numbers between CAR- andmock-transduced cells overall (FIG. 3C). CAR expression, in the absenceof target cells, induced exocytosis of lytic granules revealed by CD107aexpression (FIG. 3D), suggesting that impaired cell recovery was causedby fratricide.

Downregulation of CD7 Prevents T Cell Fratricide and does not Affect TCell Function

If the poor T-cell recovery was caused by fratricide mediated by CARbinding to CD7 expressed by the T cells, then it should improve bydownregulating CD7 prior to CAR expression. To test this prediction, arapid and practical method recently developed based on the expression ofthe anti-CD7 scFv linked to amino acid sequences containing the ERretention domains KDEL or KKMP [anti-CD7 Protein Expression Blocker(PEBL)] was applied. (FIG. 3E). These fasten the constructs to theER/Golgi, preventing secretion or membrane expression of the targetedprotein.^(39,40) 3 anti-CD7 PEBL constructs were tested and PEBL-1 wasselected PEBL-1 for the next experiments (FIGS. 3E and 3F). CD7 surfaceexpression was essentially abrogated in all T cells transduced with thisconstruct while CD7 mRNA expression was retained (FIG. 3F, FIG. 10A andFIG. 10B); in 5 experiments, 98.1%±1.5% mock-transduced T cells wereCD7+ versus 2.0%±1.7% for T cells transduced with the anti-CD7 PEBL(P<0.0001) (FIG. 3G). When the anti-CD7 CAR was expressed byelectroporation in cells with downregulated CD7, it was clearlydetectable by flow cytometry (FIG. 3H). By expressing the CAR in cellswith CD7 knock-down, T cell viability markedly improved (FIG. 3I); in 6paired experiments, viable cell recovery after CAR mRNA electroporationwas consistently superior in T cells that had been previously transducedwith the anti-CD7 PEBL (P=0.008).

After anti-CD7 PEBL transduction, the proportion of CD4 and CD8 cellswas similar to that of mock-transduced cells (FIG. 4A). Absence of CD7expression on the surface membrane did not affect T-cell survival inculture (FIG. 4B). To further probe the functional capacity of T cellstransduced with anti-CD7 PEBL, the cells were engineered to express theanti-CD19-CAR (FIG. CA). Their capacity to exert cytotoxicity, releasecytotoxic granules, and secrete IFNγ in the presence of CD19+ ALL cellswas tested. As shown in FIGS. 4D, 4E, and 4F, PEBL transduction and lackof surface CD7 did not altered CAR-mediated cell function.

Anti-CD7-41BB-CD3ζ CAR Induces Powerful Cytotoxicity Against CD7+Leukemic Cells

CD7-negative T cells were prepared using anti-CD7 PEBL, andelectroporated with the anti-CD7-41BB-CD3ζCAR mRNA. Their anti-leukemiccapacity was assessed in co-cultures with the CD7+ leukemia cell linesMOLT-4, CCRF-CEM, Jurkat, Loucy or KG1a. As shown in FIG. 5A,cytotoxicity was dramatically increased by the CAR expression. PEBL-CART cells were also highly effective against primary T-ALL cells obtainedfrom patients (FIG. 5B).

The cytotoxicity of PEBL-CAR T cells was compared to that of theresidual T cells recovered after CAR electroporation in cells nottransduced with PEBL. In 45 experiments with cells from 3 donors,cytotoxicity of the PEBL-CAR cells consistently surpassed that ofnon-PEBL T cells (FIG. 5C). The superior activity of the former cellswas also observed when comparing the expressions of CD107a (FIG. 5D),IFNγ (FIG. 11A) and TNFα (FIG. 11B). Expression of PEBL and CAR bysequential retroviral transduction also produced powerful cytotoxicityagainst patient-derived T-ALL cells (FIG. 5E) and cell lines (FIG. 12).Proliferation of anti-CD7 PEBL-CAR-T cells in the presence of CD7+target cells was much higher than that of CAR-T without CD7downregulation by PEBL(P<0.01)(FIG. 5F). Finally, the cytotoxicityexerted by anti-CD7 PEBL-CAR T cells was compared to that of T cellsexpressing an anti-CD19-41BB-CD3ζ CAR⁵ against the same target cells. Tothis end, CCRF-CEM and Jurkat cells were transduced with CD19, and alsoexpressed either CAR in cells previously transduced with anti-CD7 PEBL(FIGS. 13A and 13B). Anti-CD7 and anti-CD19 CAR T cells had similarshort- and long-term cytotoxicity (FIGS. 13C and 13D); long-termproliferative capacity in the presence of CD19+ CD7+ target cells wasslightly lower for the anti-CD7 CAR-T cells (FIG. 13E), which might beexplained by the lower expression of CD7 versus CD19 on target cells(FIG. 13B)

Anti-Leukemic Activity of Anti-CD7 PEBL-CAR T Cells in Murine Models ofT-ALL

To further gauge the anti-tumor capacity of anti-CD7 PEBL-CAR T cells,NOD/scid IL2RGnull were engrafted with CCRF-CEM cells. T cellsretrovirally transduced with anti-CD7 PEBL and anti-CD7 CAR producedconsiderable anti-leukemic effect, with a marked reduction in leukemiacell burden and a decrease in leukemia cell growth (FIGS. 6A-6C; FIGS.14A and 14B). Three weeks after leukemic cell injection, median percentCCRF-CEM cells in peripheral blood by flow cytometry was 68% for controlmice (n=5) and 67% for those who receive GFP-alone T cells (n=5), butthey were undetectable in mice treated with anti-CD7 PEBL-CAR T cells(FIG. 15A). Relapse occurring after anti-CD7 PEBL-CAR T cell treatmentwas not due to CCRF-CEM cell subsets lacking CD7; leukemic cellscontinued to express high levels of CD7 and sensitivity to anti-CD7 CARcytotoxicity remained high regardless of whether CCRF-CEM cells werederived from liver or spleen of relapsing mice or directly from theoriginal cell culture (FIG. 15B).

To test PEBL-CAR T cells against primary leukemic cells in vivo, a PDXmodel of ETP-ALL was used. The PDX model allows propagation of leukemiccells derived from a patient with ETP-ALL at diagnosis in NOD/scidIL2RGnull mice. Leukemic cells retained an immunophenotype matching thatdetermined at diagnosis, with expression of CD7, CD34, CD33, and absenceof surface CD3ζ, CD1a, CD8 and CD5 (FIG. 16); the cells were unable tosurvive and expand ex vivo, and needed to be injected in mice forpropagation. All mice had ETP-ALL in peripheral blood at the time ofCAR-T treatment (FIG. 7A). As shown in FIG. 7B, ETP-ALL cellsrepresented the majority of leukocytes in bone marrow, spleen liver andlung. After administration of PEBL-CAR T cells (2×10⁷ in one mouse,2×10⁶ in the remaining 4), leukemic cell numbers in peripheral blooddecrease dramatically, while PEBL-CAR-T cells became detectable in allmice (FIG. 7A). I n blood smears, smudge cells were prominent suggestingleukemia cell lysis (FIG. 7C). Leukemia progressed in all 5 controlmice, which were euthanized after when ETP-ALL were ≥80% of peripheralblood mononucleated cells. The mouse treated with 2×10⁷ PEBL-CAR-Tcells, died of apparent graft-versus-host disease (GvHD) 23 days afterPEBL-CAR-T cell infusion. No ETP-ALL could be detected in blood, bonemarrow, liver, spleen, lung and brain, while PEBL-CAR T cells weredetectable in all tissues (FIGS. 7D and 7E). The 4 mice treated with2×10⁶ PEBL-CAR T cells are alive, 25 (n=1) to 39 (n=3) dayspost-infusion, with no signs of GvHD.

DISCUSSION

Durable remissions in patients with B-cell leukemia and lymphoma can beachieved with CAR-T cells but effective options are lacking for patientswith T-cell malignancies. To bridge this gap, a CAR-T cell approach thatcould be rapidly translated into clinical intervention was developed anddescribed herein. CD7, a widely expressed surface T-cell marker, whichis highly stable even in T-ALL cells exposed to chemotherapy wastargeted. A second-generation anti-CD7 CAR was designed. It wasdetermined that suppression of CD7 surface expression in T cells wasessential; without it, the CAR caused severe T-cell loss, and the fullfunctional potential of CAR-T cells could not be achieved. Transductionof anti-CD7 PEBL resulted in virtually instantaneous abrogation of CD7expression. Expression of anti-CD7 CAR in such cells produced powerfulanti-leukemic activity in vitro, as well as in xenograft and PDX modelsof T-ALL. Thus, by using this strategy, large numbers of CAR-T cellswere rapidly generated and were used to exert robust and specificcytotoxicity against T-cell malignancies, including one of the mostaggressive forms, ETP-ALL.

The PEBL technology as described herein to downregulate endogenous CD7is based on the use of a scFv directed against the targeted antigencoupled with an ER/Golgi-retention motif. In this way, any newlysynthesized CD7 remains anchored in the ER and/or Golgi, and its surfaceexpression is prevented. This method was remarkably effective indownregulating CD7 and suppressing CAR-mediated fratricide. Importantly,intracellular retention of CD7 did not alter T-cell function and allowednormal expansion, cytokine secretion, and cytotoxicity. This isconsistent with results of studies with CD7-deficient mice which showednormal lymphocyte populations in lymphoid tissues.^(41,42) Analternative approach to downregulate CD7 would be to apply gene editingmethods, such as meganucleases, TALEN, or CRISPR/Cas9.⁴³ To this end, arecent study reported an anti-CD7 CAR which was expressed in T cellswith CD7 gene deletion by CRISPR/Cas.^(9,44) Besides differences inco-stimulatory molecules (the CAR described herein has 4-1BB instead ofCD28) which may have clinical impact,^(45,46) the high specificity andpractical nature of the PEBL strategy make it particularly attractivefor current clinical use. This method requires a simple transductionwith the same viral vector carrying the CAR, either as two sequentialtransductions or a single transduction with a bicistronic vectorcarrying both constructs. It fits well with established clinical-gradecell manufacturing processes, and does not raise possible regulatoryconcerns associated with off-target activity.^(47,48)

CD7 is a hallmark molecule for early T-cell differentiation; it isnearly universally expressed in T-ALL, and among normal cells, itsexpression is limited to T cells.^(19,29-32) In a clinical study with ananti-CD7-ricin-A-chain immunotoxin in patients with T-cell lymphoma, thedose-limiting toxicity was vascular leak syndrome, a side-effect seenwith other toxin-conjugates; no binding of anti-CD7 was found inendothelial cells of various tissues.⁴⁹ Nevertheless, transientexpression of the CAR by mRNA electroporation might be considered inearly studies assessing potential for acute toxicities of anti-CD7PEBL-CAR T cells. A concern of anti-CD7 CAR therapy is the depletion ofnormal T cells by the infused cells, leading to immunodeficiency. Onecan envisage the initial application of this technology as a means toreduce MRD in patients with high-risk T-ALL, therefore maximizing thesuccess of allogeneic hematopoietic stem cell transplantation.⁵⁰ In suchinstances, anti-CD7 CAR T cells would be eliminated by the transplantconditioning and the T-cell compartment reconstituted from donor stemcells. Outside the transplant setting, “suicide genes” could beactivated once leukemia eradication has been achieved.⁵¹ Ultimately,this may not be an issue, as the infused anti-CD7 T cells (which retaintheir endogenous CD3/TCR complex) might reconstitute a sufficiently wideT-cell repertoire. To this end, it should be noted that subsets of CD4memory and CD8 effector T cells in human blood lymphocyte which do notexpress CD7 have been described,^(52,53) and that T-ALL cells expressCD7 at higher levels than normal T cells. Thus, CD7-dim subsets mighthelp to repopulate the T-cell repertoire even after CD7-directedtherapy.

The standard treatment of T-ALL mainly relies on intensive chemotherapyplus hematopoietic stem cell transplant for patients with high-riskdisease. Results are far from satisfactory and have considerablemorbidity and mortality.^(54,55) The findings presented herein suggestthe infusion of anti-CD7 PEBL-CAR T cells could significantly enhance,or perhaps replace, existing chemotherapy- and transplant-basedstrategies. Conceivably, CAR expression together with downregulation ofthe targeted antigen in T cells should also be applicable to other Tcell markers, such as CD3ζ, CD2, and CD5 whose expression is prevalentin T-cell lymphoproliferative neoplasms. Because a fraction of high-riskacute myeloid leukemia cases express CD7,^(19,30,56) testing thepotential of anti-CD7 CAR-T cells for this leukemia subtype is alsowarranted.

REFERENCES

-   1. Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and    targeting of cytotoxic lymphocytes through chimeric single chains    consisting of antibody-binding domains and the gamma or zeta    subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad    Sci USA 1993; 90(2): 720-724.-   2. Geiger T L, Leitenberg D, Flavell R A. The TCR zeta-chain    immunoreceptor tyrosine-based activation motifs are sufficient for    the activation and differentiation of primary T lymphocytes. J    Immunol 1999; 162(10):5931-5939.-   3. Brentjens R J, Latouche J B, Santos E, et al. Eradication of    systemic B-cell tumors by genetically targeted human T lymphocytes    co-stimulated by CD80 and interleukin-15. Nat Med 2003;    9(3):279-286.-   4. Cooper L J, Topp M S, Serrano L M, et al. T-cell clones can be    rendered specific for CD19: toward the selective augmentation of the    graft-versus-B-lineage leukemia effect. Blood 2003;    101(4):1637-1644.-   5. Imai C, Mihara K, Andreansky M, Nicholson I C, Pui C H,    Campana D. Chimeric receptors with 4-1BB signaling capacity provoke    potent cytotoxicity against acute lymphoblastic leukemia. Leukemia    2004; 18:676-684.-   6. Rosenberg S A, Restifo N P. Adoptive cell transfer as    personalized immunotherapy for human cancer. Science 2015;    348(6230):62-68.-   7. Brentjens R J, Santos E, Nikhamin Y, et al. Genetically targeted    T cells eradicate systemic acute lymphoblastic leukemia xenografts.    Clin Cancer Res 2007; 13(18 Pt 1):5426-5435.-   8. Haso W, Lee D W, Shah N N, et al. Anti-CD22-chimeric antigen    receptors targeting B-cell precursor acute lymphoblastic leukemia.    Blood 2013; 121(7):1165-1174.-   9. Nadler L M, Anderson K C, Marti G, et al. B4, a human B    lymphocyte-associated antigen expressed on normal,    mitogen-activated, and malignant B lymphocytes. J Immunol 1983;    131(1):244-250.-   10. Campana D, Janossy G, Bofill M, et al. Human B cell    development. I. Phenotypic differences of B lymphocytes in the bone    marrow and peripheral lymphoid tissue. J Immunol 1985;    134(3):1524-1530.-   11. Porter D L, Levine B L, Kalos M, Bagg A, June C H. Chimeric    antigen receptor-modified T cells in chronic lymphoid leukemia. N    Engl J Med 2011; 365(8):725-733.-   12. Grupp S A, Kalos M, Barrett D, et al. Chimeric antigen    receptor-modified T cells for acute lymphoid leukemia. N Engl J Med    2013; 368(16):1509-1518.-   13. Till B G, Jensen M C, Wang J, et al. CD20-specific adoptive    immunotherapy for lymphoma using a chimeric antigen receptor with    both CD28 and 4-1BB domains: pilot clinical trial results. Blood    2012; 119(17):3940-3950.-   14. Kochenderfer J N, Dudley M E, Feldman S A, et al. B-cell    depletion and remissions of malignancy along with    cytokine-associated toxicity in a clinical trial of anti-CD19    chimeric-antigen-receptor-transduced T cells. Blood 2012;    119(12):2709-2720.-   15. Davila M L, Riviere I, Wang X, et al. Efficacy and Toxicity    Management of 19-28z CAR T Cell Therapy in B Cell Acute    Lymphoblastic Leukemia. Sci Transl Med 2014; 6(224): 224ra225.-   16. Maude S L, Frey N, Shaw P A, et al. Chimeric antigen receptor T    cells for sustained remissions in leukemia. N Engl J Med 2014;    371(16):1507-1517.-   17. Lee D W, Kochenderfer J N, Stetler-Stevenson M, et al. T cells    expressing CD19 chimeric antigen receptors for acute lymphoblastic    leukaemia in children and young adults: a phase 1 dose-escalation    trial. Lancet 2015; 385(9967):517-528.-   18. Turtle C J, Hanafi L A, Berger C, et al. CD19 CAR-T cells of    defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin    Invest 2016; 126(6):2123-2138.-   19. Coustan-Smith E, Mullighan C G, Onciu M, et al. Early T-cell    precursor leukaemia: a subtype of very high-risk acute lymphoblastic    leukaemia. Lancet Oncol 2009; 10(2): 147-156.-   20. Zhang J, Ding L, Holmfeldt L, et al. The genetic basis of early    T-cell precursor acute lymphoblastic leukaemia. Nature 2012;    481(7380):157-163.-   21. Inukai T, Kiyokawa N, Campana D, et al. Clinical significance of    early T-cell precursor acute lymphoblastic leukaemia: results of the    Tokyo Children's Cancer Study Group Study L99-15. Br J Haematol    2012; 156(3):358-365.-   22. Neumann M, Heesch S, Gokbuget N, et al. Clinical and molecular    characterization of early T-cell precursor leukemia: a high-risk    subgroup in adult T-ALL with a high frequency of FLT3 mutations.    Blood Cancer J 2012; 2(1):e55.-   23. Jain N, Lamb A V, O'Brien S, et al. Early T-cell precursor acute    lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) in adolescents and    adults: a high-risk subtype. Blood 2016; 127(15): 1863-1869.-   24. Marks D I, Rowntree C. Management of adults with T-cell    lymphoblastic leukemia. Blood 2017; 129(9):1134-1142.-   25. Campana D, Pui C H. Minimal residual disease-guided therapy in    childhood acute lymphoblastic leukemia. Blood 2017;    129(14):1913-1918.-   26. Coustan-Smith E, Sancho J, Hancock M L, et al. Use of peripheral    blood instead of bone marrow to monitor residual disease in children    with acute lymphoblastic leukemia. Blood 2002; 100:2399-2402.-   27. Mihara K, Yanagihara K, Takigahira M, et al. Activated    T-cell-mediated immunotherapy with a chimeric receptor against CD38    in B-cell non-Hodgkin lymphoma. J Immunother 2009; 32(7):737-743.-   28. Mamonkin M, Rouce R H, Tashiro H, Brenner M K. A T-cell-directed    chimeric antigen receptor for the selective treatment of T-cell    malignancies. Blood 2015; 126(8):983-992.-   29. Haynes B F, Eisenbarth G S, Fauci A S. Human lymphocyte    antigens: production of a monoclonal antibody that defines    functional thymus-derived lymphocyte subsets. Proc Natl Acad Sci USA    1979; 76(11):5829-5833.-   30. Vodinelich L, Tax W, Bai Y, Pegram S, Capel P, Greaves M F. A    monoclonal antibody (WT1) for detecting leukemias of T-cell    precursors (T-ALL). Blood 1983; 62(5):1108-1113.-   31. Janossy G, Coustan-Smith E, Campana D. The reliability of    cytoplasmic CD3ζ and CD22 antigen expression in the immunodiagnosis    of acute leukemia: a study of 500 cases. Leukemia 1989;    3(3):170-181.-   32. Yeoh E J, Ross M E, Shurtleff S A, et al. Classification,    subtype discovery, and prediction of outcome in pediatric acute    lymphoblastic leukemia by gene expression profiling. Cancer Cell    2002; 1:133-143.-   33. Manabe A, Coustan-Smith E, Kumagai M, et al. Interleukin-4    induces programmed cell death (apoptosis) in cases of high-risk    acute lymphoblastic leukemia. Blood 1994; 83(7): 1731-1737.-   34. Peipp M, Kupers H, Saul D, et al. A recombinant CD7-specific    single-chain immunotoxin is a potent inducer of apoptosis in acute    leukemic T cells. Cancer Res 2002; 62(10):2848-2855.-   35. Kudo K, Imai C, Lorenzini P, et al. T lymphocytes expressing a    CD16 signaling receptor exert antibody-dependent cancer cell    killing. Cancer Res 2014; 74(1):93-103.-   36. Shimasaki N, Fujisaki H, Cho D, et al. A clinically adaptable    method to enhance the cytotoxicity of natural killer cells against    B-cell malignancies. Cytotherapy. 2012; 14(7):830-40.-   37. Shimasaki N, Campana D. Natural killer cell reprogramming with    chimeric immune receptors. Methods Mol Biol 2013; 969:203-220.-   38. Chang Y H, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D.    A Chimeric Receptor with NKG2D Specificity Enhances Natural Killer    Cell Activation and Killing of Tumor Cells. Cancer Res 2013;    73(6):1777-1786.-   39. Munro S, Pelham H R. A C-terminal signal prevents secretion of    luminal E R proteins. Cell 1987; 48(5):899-907.-   40. Jackson M R, Nilsson T, Peterson P A. Identification of a    consensus motif for retention of transmembrane proteins in the    endoplasmic reticulum. EMBO J 1990; 9(10):3153-3162.-   41. Bonilla F A, Kokron C M, Swinton P, Geha R S. Targeted gene    disruption of murine CD7. Int Immunol 1997; 9(12):1875-1883.-   42. Lee D M, Staats H F, Sundy J S, et al. Immunologic    characterization of CD7-deficient mice. J Immunol 1998;    160(12):5749-5756.-   43. Boettcher M, McManus M T. Choosing the right tool for the job:    RNAi, TALEN, or CRISPR. Mol Cell 2015; 58(4):575-585.-   44. Gomes-Silva D, Srinivasan M, Sharma S, et al. CD7-edited T cells    expressing a CD7-specific CAR for the therapy of T-cell    malignancies. Blood. 2017 May 24. pii: blood-2017-01-761320.-   45. Campana D, Schwarz H, Imai C. 4-1BB chimeric antigen receptors.    Cancer J 2014; 20(2):134-140.-   46. Zhao Z, Condomines M, van der Stegen S J, et al. Structural    Design of Engineered Costimulation Determines Tumor Rejection    Kinetics and Persistence of CAR T Cells. Cancer Cell 2015;    28(4):415-428.-   47. Tsai S Q, Joung J K. Defining and improving the genome-wide    specificities of CRISPR-Cas9 nucleases. Nat Rev Genet 2016;    17(5):300-312.-   48. Cameron P, Fuller C K, Donohoue P D, et al. Mapping the genomic    landscape of CRISPR-Cas9 cleavage. Nat Methods 2017; 14(6):600-606.-   49. Frankel A E, Laver J H, Willingham M C, Burns L J, Kersey J H,    Vallera D A. Therapy of patients with T-cell lymphomas and leukemias    using an anti-CD7 monoclonal antibody-ricin A chain immunotoxin.    Leuk Lymphoma 1997;26(3-4):287-298.-   50. Leung W, Pui C H, Coustan-Smith E, et al. Detectable minimal    residual disease before hematopoietic cell transplantation is    prognostic but does not preclude cure for children with    very-high-risk leukemia. Blood 2012; 120(2):468-72.-   51. Straathof K C, Spencer D M, Sutton R E, Rooney C M. Suicide    genes as safety switches in T lymphocytes. Cytotherapy 2003;    5(3):227-230.-   52. Reinhold U, Abken H, Kukel S, et al. CD7− T cells represent a    subset of normal human blood lymphocytes. J Immunol 1993;    150(5):2081-2089.-   53. Aandahl E M, Sandberg J K, Beckerman K P, Tasken K, Moretto W J,    Nixon D F. CD7 is a differentiation marker that identifies multiple    CD8 T cell effector subsets. J Immunol 2003; 170(5):2349-2355.-   54. Raetz E A, Teachey D T. T-cell acute lymphoblastic leukemia.    Hematology Am Soc Hematol Educ Program 2016; 2016(1):580-588.-   55. Jabbour E, O'Brien S, Konopleva M, Kantarjian H. New insights    into the pathophysiology and therapy of adult acute lymphoblastic    leukemia. Cancer 2015; 121(15):2517-2528.-   56. Kita K, Miwa H, Nakase K, et al. Clinical importance of CD7    expression in acute myelocytic leukemia. The Japan Cooperative Group    of Leukemia/Lymphoma. Blood 1993; 81(9):2399-2405.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. An expression vector comprising: a) a firstnucleic acid comprising a nucleotide sequence encoding a target-bindingmolecule linked to a localizing domain, wherein said target-bindingmolecule comprises a first antibody that binds to CD7; b) a secondnucleic acid comprises a nucleotide sequence encoding the CD7 CAR,wherein said CD7 CAR comprises a second antibody that binds to CD7;wherein an amino acid sequence of the first antibody and an amino acidsequence of the second antibody are at least 80% identical.
 2. Theexpression vector of claim 1, wherein the amino acid sequence of thefirst antibody and the amino acid sequence of the second antibody are atleast 90% identical.
 3. The expression vector of claim 1, wherein aminoacid sequences of CDR regions of the first antibody and amino acidsequences of CDR regions of the second antibody are at least 80%identical.
 4. The expression vector of claim 1, wherein the firstantibody comprises a heavy chain variable domain having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:14, or SEQ ID NO: 16 and a light chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO:2, SEQ IDNO: 15, or SEQ ID NO:
 17. 5. The expression vector of claim 1, whereinthe second antibody comprises a heavy chain variable domain having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:1,SEQ ID NO: 14, or SEQ ID NO: 16 and a light chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:2, SEQ ID NO: 15, or SEQ ID NO:
 17. 6. The expression vector of claim1, wherein the first nucleic acid comprises a nucleotide sequence havingat least 90% sequence identity to SEQ ID NO: 23 and a nucleotidesequence having at least 90% sequence identity to SEQ ID NO: 24
 7. Theexpression vector of claim 1, wherein the localization domain comprisesan endoplasmic reticulum (ER) retention sequence, a Golgi retentionsequence, a proteasome localizing sequence, or a transmembrane domain.8. The expression vector of claim 1, wherein the CD7 CAR comprises a4-1BB intracellular signaling domain, and a CD3ζ intracellular signalingdomain.
 9. An engineered immune cell comprising an expression vector ofclaim
 1. 10. The engineered immune cell of claim 9, wherein theengineered immune cell is a T cell.
 11. A method for treating CD7positive cancer in a patient in need thereof, comprising administering atherapeutically effective amount of an engineered immune cell, whereinsaid engineered immune cell comprises i) a chimeric antigen receptor(CAR) expressed on the surface of the immune cell, wherein the CARspecifically binds to CD7, and ii) a CD7 binding domain linked to anintracellular localization domain; and thereby treating the CD7 positivecancer.
 12. The method of claim 11, wherein the CD7 positive cancer is aT cell malignancy.
 13. The method of claim 11, wherein the engineeredimmune cell is a T cell.
 14. The method of claim 11, wherein the bindingof endogenous CD7 to the CD7 binding domain reduces surface expressionof the endogenous CD7 in the engineered immune cell, thereby increasingthe effectiveness of the engineered immune cell against CD7 positivecancer cells.
 15. The method of claim 11, wherein the CAR comprises afirst scFv domain and the CD7 binding domain comprises a second scFvdomain, wherein amino acid sequence of the first scFv domain and aminoacid sequence of the second scFv domain are at least 80% identical. 16.The method of claim 11, wherein each of first and second scFvs comprisesa heavy chain variable domain having at least 90% sequence identity tothe amino acid sequence of SEQ ID NO:1, SEQ ID NO: 14, or SEQ ID NO: 16and a light chain variable domain having at least 90% sequence identityto the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 15, or SEQ ID NO:17.
 17. A method of producing the engineered immune cell expressing achimeric antigenic receptor (CAR) targeting a cell surface protein, themethod comprising: i) introducing into an immune cell: a) a firstnucleic acid comprising a nucleotide sequence encoding a target-bindingmolecule linked to a localizing domain, wherein said target-bindingmolecule is a first antibody that specifically binds to the cell surfaceprotein; b) a second nucleic acid comprises a nucleotide sequenceencoding the CAR, wherein said CAR comprises a second antibody thatspecifically binds to the cell surface protein; and ii) culturing theengineered immune cell comprising said target-binding molecule linked tosaid localizing domain and said CAR, thereby producing said engineeredimmune cell; wherein expression of the target-binding molecule preventsfratricide of the engineered immune cell during step ii).
 18. The methodof claim 17, wherein the immune cell is a T cell.
 19. The method ofclaim 17, wherein the cell surface protein is CD7.
 20. The method ofclaim 17, wherein the CAR comprises a first scFv domain and the targetbinding molecule comprises a second scFv domain, wherein amino acidsequence of the first scFv domain and amino acid sequence of the secondscFv domain are at least 80% identical.
 21. The method of claim 17,wherein the introduction of the first and second nucleic acids does notaffect expansion or function of the engineered immune cell.